Following the Alabama Supreme Court's ruling, the  University of Alabama at Birmingham  announced that it was pausing  in vitro fertilization  treatments. [ 17 ]  Two other IVF clinics also paused treatments following the ruling. [ 8 ]   Alabama Attorney General   Steve Marshall  that he "has no intention of using the recent Alabama Supreme Court decision as a basis for prosecuting IVF families or providers." [ 18 ]  The next day, an  explosive device  was detonated outside of Marshall's office in Montgomery, although no one was harmed in the incident. [ 19 ]


State lawmakers from both political parties reacted the same week to protect IVF. A bill with bipartisan support and backing from Alabama's governor  Kay Ivey  was introduced that "would provide that any fertilized human egg or human embryo that exists outside of a human uterus is not considered an unborn child or human being for any purpose under state law" and allow IVF clinics to proceed as normal. [ 20 ] [ 21 ]  On February 29, 2024, the two houses of the Alabama legislature passed similar bills, stating "no action, suit, or criminal prosecution for the damage to or death of an embryo shall be brought or maintained against any individual or entity when providing or receiving goods or services related to in vitro fertilization." [ 22 ]  Ivey signed the law on March 6, 2024, with two of the IVF clinics stating they would immediately resume services. [ 23 ]


At the federal level, a bill titled the Access to Family Building Act was introduced in Congress in January 2024, which would both create a statutory right to IVF and override all conflicting federal, state, or local regulations, whether adopted before, on, or after its enactment, including the  Religious Freedom Restoration Act of 1993 . [ 24 ]  The bill's co-sponsor Senator  Tammy Duckworth  urged action on the bill in light of the Alabama court ruling. [ 20 ]  However, Senate Republicans blocked the bill during voting on February 28, 2024. [ 25 ]


According to David Schultz, professor of law at University of Minnesota, this ruling cannot be appealed to the U.S. Supreme Court, as the decision is solely an interpretation of Alabama state law. Schultz also argues that this ruling would have been possible prior to the overturning of federal abortion protection in the 2022  Dobbs  decision, because it does not directly relate to abortion. [ 26 ]


The Alabama case was seen to have a potentially significant impact on the  2024 United States presidential election , following the U.S. Supreme Court decision to overturn  Roe v. Wade  in the 2022 case  Dobbs v. Jackson Women's Health Organization . The  Democratic party  started to immediately add the issue of protecting IVF treatments to its campaign platform in addition to its fight to protect abortion and reproductive services. President  Joe Biden  condemned the ruling, stating it was only possible due to the decision from  Dobbs  and that "the disregard for women's ability to make these decisions for themselves and their families is outrageous and unacceptable." [ 27 ] [ 28 ] [ 4 ]  Vice President  Kamala Harris  said, "On the one hand, the proponents are saying that an individual doesn't have a right to end an unwanted pregnancy and, on the other hand, the individual does not have the right to start a family." [ 28 ] [ 4 ]


Some members of the  Republican party , already trying to push their stance on abortion to the background so as to draw votes from women and swing voters, were critical of the Alabama Supreme Court decision since it left no carve out to protect IVF. [ 28 ] [ 29 ]  Former president  Donald Trump , running in 2024 for a  second term as president , stated that access to IVF and fertility treatments should remain legal. [ 30 ]   Nikki Haley , another Republican candidate, initially stated that "embryos are babies," but later said she would need to review the law, stating, "We don't want fertility treatments to shut down." [ 28 ]  New Hampshire Governor  Chris Sununu  and Tennessee Governor  Bill Lee  also spoke out against the decision. [ 27 ]  Other Republican leaders in the U.S. House of Representatives praised the decision, having previously backed a bill that would codify that life begins at conception. [ 31 ]


Chief Justice Tom Parker's concurring opinion rested heavily on Christian principles, leading to concern about his overall approach to judiciary duties. The same day of the decision, an interview with Parker was released where he expressed his beliefs in  Christian nationalism  and support of the  Seven Mountain Mandate . Parker said in this interview, "God created government, and the fact that we have let it go into the possession of others, it's heartbreaking. That's why he is calling and equipping people to step back into these mountains right now." [ 32 ]


Parker's comments, in addition to his judicial opinion, have led to concern about the push of Christian nationalism by conservative politicians and the Republican party, and the attempted weakening of the  separation of church and state . [ 32 ] [ 33 ] [ 34 ]


Miriam Friedman Menkin  (8 August 1901 – June 8, 1992), née  Miriam Friedman , was an American scientist who was most famous for her  in vitro fertilization  (IVF) research with  John Rock . In February 1944, she became the first person to conceive human life outside of the body. [ 1 ]


Menkin was born on August 8, 1901, in  Riga ,  Latvia , and died at the age of 90 on June 8, 1992, in Boston, Massachusetts . Two years later, her family relocated to the United States. Her father was a successful doctor in New York City, allowing her family to live securely. [ 2 ]  In 1922, Menkin graduated from  Cornell University  with an undergraduate degree in histology and comparative anatomy. [ 3 ] [ 4 ]  She attended  Columbia University  for her graduate program and earned a master's degree in genetics only one year after graduating from Cornell. [ 2 ]  She taught biology and physiology for a short period while setting her sights on medical school. However, women were rarely admitted to medical school at the time and she was not accepted. [ 2 ] [ 4 ]   Menkin married Valy Menkin, a  Harvard  medical student, in 1924. She still intended to earn a Ph.D. in biology, but she needed to provide financial support while her husband finished medical school. Thus, she obtained a second undergraduate degree in secretarial studies from  Simmons College . [ 3 ]  Menkin ended up finishing the Harvard Ph.D. requirements two separate times but did not receive a degree because she could not afford the course fees. [ 5 ]


Menkin and Valy had two children: a son named Gabriel and a daughter named Lucy. [ 2 ] [ 4 ]


Following a few years of work as a secretary, Menkin served as a pathology research fellow at  Harvard Medical School  from 1930 to 1935. [ 3 ]  She was then offered a job as a laboratory technician for  Gregory Pincus  at Harvard. While working for Pincus, Menkin was tasked with preparing extracts designed to superovulate rabbits in Pincus' quest to create "fatherless" rabbits. [ 3 ]  Pincus lost his tenure at Harvard in 1937, which left her without a job. She worked in the state laboratories of Massachusetts for a year and then applied for a research position with John Rock, a fertility doctor, at the Free Hospital for Women (now part of  Brigham and Women's Hospital ) in Boston. [ 2 ]  Pincus' rabbit experiments had been a factor in Rock's decision to start IVF research, and Menkin's role in the experiments caught Rock's eye. [ 2 ] [ 4 ]  Rock soon hired Menkin and they set out to determine the exact time at which  ovulation  would occur. Rock had come up with the idea but had not made any meaningful progress in laboratory research before hiring Menkin. [ 2 ]  He did not have an advanced understanding of the technical aspects of egg  fertilization [ 3 ]  and hired Menkin to oversee all of the laboratory work.


Menkin began the IVF experiments in March 1938. [ 1 ]  Rock and Menkin requested that the women participating in the study, who were scheduled to undergo  hysterectomies , have unprotected sex prior to the surgery. [ 6 ]  They elected to carry out the surgical procedures just before the patient would ovulate, which gave them many suitable ova for the study. [ 7 ]  Over the course of the study, Menkin followed a fairly steady weekly schedule: find eggs on Tuesday, add sperm on Wednesday, pray on Thursday, and observe the outcome using a microscope on Friday. [ 1 ] [ 4 ]  Menkin called herself the 'egg chaser' because she would take the ovarian tissue from an operation and ran up three flights of stairs to the lab to look for eggs. She would then try to fertilize the eggs that she found with the 'left over' sperm. February 6, she finally fertilized her first egg. She made variations to the procedure every so often, altering the conditions the eggs were kept in and the length and concentration of the sperm samples. [ 8 ]  Menkin would find the unfertilized eggs in the ovaries that Rock removed from the patients, put the eggs in the solution, and then culture them before adding sperm. [ 1 ]  In the first 6 years of the study, Menkin tried many different strategies but did not achieve IVF. [ 1 ] [ 7 ] [ 8 ]  On February 3, 1944, she obtained an egg from a woman whose cervix and uterus  prolapsed  following the birth of four children. [ 2 ]  Menkin's standard protocol was to wash the sperm sample 3 times in solution [ 3 ]  and let the egg and sperm interact for 30 minutes. [ 2 ]  However, she had stayed up with her newborn daughter the night before, and on that day she mistakenly washed the sperm sample only once, used a more concentrated sample, and allowed one hour of interaction. [ 2 ] [ 3 ] [ 4 ] [ 8 ]  The following Friday morning (February 6, 1944), Menkin found that  cell cleavage  had begun, [ 8 ]  which indicated that a fertilized egg had formed. Menkin neglected to take an immediate photograph of the discovery, and she could not find the egg when she finally went to get a picture. [ 3 ]  She was able to fertilize and photograph three more eggs, involving all three factors that she had altered before the original discovery. [ 3 ]  Rock and Menkin achieved two and three cell development in their successful fertilizations. [ 8 ] [ 9 ]  After the additional eggs were fertilized successfully, Rock and Menkin elected to publish their work in a brief report.  Science  magazine published their findings in the article “ In Vitro  Fertilization and Cleavage of Human Ovarian Eggs" on August 4, 1944. [ 8 ]  The  Associated Press ,  The New York Times , and  Time  magazine ran accounts of the discovery in the following days. [ 7 ]  In March 1938, Menkin began his IVF research. The public perceived Rock and Menkin's discovery as a positive step toward assisting infertile couples in having children.


Rock and Menkin's discovery was largely viewed by the public as a positive breakthrough that would help infertile couples have children. [ 3 ]  However, some critics saw it as playing God and interfering with the laws of nature, and because Rock and Menkin did not create a physical baby, others questioned if they had achieved in vitro fertilization at all. [ 3 ]  Rock and Menkin did not attempt to transfer their fertilized eggs into a woman's body [ 1 ]  after their achievement because it was not the goal of their study. [ 8 ]  The difficult task of transforming a multi-cell fertilized egg, like the one Menkin and Rock had created, into an embryo that could grow inside a woman's uterus was not successful until the birth of  Louise Brown  in 1978. [ 9 ]


At the time  Science  ran the article, Valy had recently been fired by Harvard and the Menkins had been forced to relocate to North Carolina. Menkin was able to stay in touch with Rock and work on additional publications. The full report of the discovery, in which Menkin was listed as the lead author, was finally published in 1948 with the same title, "In Vitro Fertilization and Cleavage of Human Ovarian Eggs", in the  American Journal of Obstetrics and Gynecology . [ 9 ]  In the years before this, Menkin sought to continue IVF research on her own, but her opportunities were limited and unpaid.


Menkin divorced her husband in 1949. [ 2 ]  Supporting and caring for her daughter, Lucy, who had  epilepsy , further limited her opportunities to do laboratory research. In 1950 she returned to Boston to enroll her in a special school and accepted an offer to return to Rock's laboratory. [ 2 ] [ 4 ]  IVF research had ceased in Rock's lab since Menkin had departed, as he had been unable to find another scientist with the technical skill to continue Menkin's work. [ 3 ]  She attempted to revitalize it when she returned, but by that time Rock had shifted his primary focus to research on the  birth control pill  and Menkin was only able to work on IVF for a short time. She continued to work in Rock's lab, assisting him with his research and publications on contraception, [ 4 ] [ 5 ]  but never received another opportunity to pursue IVF. Menkin died on June 8, 1992, in  Boston, Massachusetts .


Rock, John; Menkin, Miriam F. (1944). "In Vitro Fertilization and Cleavage of Human Ovarian Eggs".  Science .  100  (2588):  105– 107.  Bibcode : 1944Sci...100..105R .  doi : 10.1126/science.100.2588.105 .  JSTOR   1672795 .  PMID   17788930 .


Menkin, Miriam F.; Rock, John (1948). "In Vitro Fertilization and Cleavage of Human Ovarian Eggs".  American Journal of Obstetrics and Gynecology .  55  (3):  440– 452.  doi : 10.1016/S0002-9378(15)32963-X .  PMID   18903892 .


Rock, John; García, Celso-Ramón; Menkin, Miriam F. (1959).  "A Theory of Menstruation" .  Annals of the New York Academy of Sciences .  75  (2):  831– 839.  Bibcode : 1959NYASA..75..831R .  doi : 10.1111/j.1749-6632.1959.tb44594.x .  ISSN   1749-6632 .  PMID   13854539 .  S2CID   44933674 .


Hertig, Arthur T.; Adams, Eleanor C.; McKay, Donald G.; Rock, John; Mulligan, William J.; Menkin, Miriam F. (1958-11-01). "A thirteen-day human ovum studied histochemically".  American Journal of Obstetrics and Gynecology .  76  (5):  1025– 1043.  doi : 10.1016/0002-9378(58)90185-6 .  PMID   13583048 .


Dewan, Edmond M.; Menkin, Miriam F.; Rock, John (1978-05-01). "Effect of Photic Stimulation on the Human Menstrual Cycle".  Photochemistry and Photobiology .  27  (5):  581– 585.  doi : 10.1111/j.1751-1097.1978.tb07649.x .  ISSN   1751-1097 .  PMID   674392 .  S2CID   19957895 .


Rock, John; Menkin, Miriam F. (1942-10-08). "Stilbestrol".  New England Journal of Medicine .  227  (15):  552– 556.  doi : 10.1056/NEJM194210082271505 .  ISSN   0028-4793 .  PMID   14461256 .


Morphokinetics  ( ‘morpho’’  form/shape [ 1 ]   and ‘kinetics’  movement [ 2 ] ) refers to time specific morphological changes during embryo development providing dynamic information on a fertilized egg.  The detailed information eases morphological selection of embryos with high implantation potential to be used in  In-Vitro Fertilisation  treatment.


The traditional method for selection of embryos to be used for embryo transfer in fertility treatments such as IVF,  involves assessing the  morphology  of a fertilized egg at crucial time points under a microscope.


The incorporation of  time lapse imaging  tools in an incubator is a novel technology that allows images of embryo development to be captured frequently. [ 3 ] [ 4 ]


This together with softwares [ 5 ]  allows embryologists and clinicians to interpret the embryo development information captured and select embryos for transfer in IVF treatment.


Research  [ 6 ]  for an  algorithm  to select a single embryo that would lead to the best clinical pregnancy outcome is underway.


Time-Lapse incubators  can be used for any patient, but they are especially indicated for:


-Patients with previous cycles of  implantation failure .


-Patients with recurrent miscarriages.


-Patients with many oocytes who wish to transfer a single embryo, since it improves their selection.
 [ 7 ]


Morphology  of the  embryo  is studied in scientific research and often marked by stages.


For further details of the processes that occur within each stage, see  Human embryogenesis .


1.   Fertilization


After the egg is fertilized by the sperm, a  zygote  (single diploid cell) is formed. At this stage, it should have two  pronuclei , one of each derived from the egg and the sperm cell respectively and two tiny cells called  polar bodies .


2.   Cleavage


This begins when the zygote divides into two cells via  mitosis . The division continues such that each cell divides into another two cells, which results in a multiplying effect.


3.    Morula


The ball of cells formed after the multiple divisions is called a  morula . It consists of about 16-32 cells in a ball within a translucent, elastic layer called the  zona pellucida . It will then undergo compaction which is a process where the cells bind firmly together and continue to develop into a blastocyst. [ 8 ]


4.   Blastocyst


Blastulation  is marked by the appearance of a fluid-filled cavity ( blastocoel ) surrounded by a single layer of cells called the  trophectoderm  and the  inner cell mass . The fetus is developed from the inner cell mass while the placenta is derived from the trophectoderm. [ 9 ]


Morphokinetics has the potential advantage over standard morphological evaluation as it does not use a static form of observation to evaluate a highly dynamic process. [ 5 ]


High resolution images obtained at frequent time points provides greater detail of the events involved in embryo development. [ 10 ]  This higher degree of detail and parameters used to assess embryo viability reduces the biased variability in embryo selection by an embryologist, allowing for a more standardised method of embryo quality evaluation and selection for implantation. [ 10 ]  This also provides a quantifiable method for analysis rather than just qualitative. [ 5 ]


Clinicians are given more time to evaluate an image rather than a time pressured evaluation of an embryo which must be quickly put back into incubation; resulting in a reduced degree of human error. [ 11 ]


Potential damage to otherwise viable embryos is also reduced due to minimized fluctuation of optimal culture conditions . These factors such as pH and temperature, if altered, have been shown to influence preimplantation development of a blastocyst. [ 12 ]


Both the mother and child may benefit from this new method of IVF treatment. Transfer of the single, highest potential embryo into the mother reduces the likelihood of multiple gestations, which limits future complications such as preterm labour. [ 11 ]


This method reduces time spent and consequently lowering overall costs. [ 12 ]


Greater understanding and evidence for embryo morphokinetics can help improve current knowledge of embryo development and factors influencing successful clinical pregnancy outcome. [ 5 ]


Although morphokinetics has its advantage in terms of selecting embryos for implantation, the technology is unable to deselect all abnormal embryos.


There is also a struggle to find a universal algorithm as attempts to validate published algorithms have proved unsuccessful. More work needs to be done in the scientific community, working as a cohesive unit to define the best possible algorithm that could lead to a single successful pregnancy. [ 6 ]


The benefits of using morphokinetics in terms of improving clinical outcomes of IVF is still very unclear . [ 9 ]  A meta-analysis published in 2017, supported the idea of clinical benefits of time-lapse culture with morphokinetic embryo selection in IVF, reporting reduced early pregnancy loss, higher ongoing pregnancy and higher live birth rates. [ 13 ]  However, the studies included in the meta analysis were carried out in selected populations and were of dubious quality, meaning we can not rely on this as conclusive evidence that IVF outcomes are improved by using morphokinetic embryo selection and time-lapse technology instead of standard morphological evaluation. [ 13 ]


Apart from this, there is limited reviewed evidence to support the clinical benefits of morphokinetics and so robust prospective studies reporting clinical outcomes must be carried out before it can be concluded that morphokinetics when combined with time-lapse technology has the ability to improve clinical outcomes of IVF. [ 13 ]


Natural Cycle In Vitro Fertilization  (IVF) is an assisted reproductive technique designed to closely mimic a woman's natural menstrual cycle. In traditional IVF, a woman's ovaries are stimulated with fertility medications to produce multiple eggs, which are then retrieved and fertilized outside the body. A natural cycle IVF, on the other hand, works with the woman's natural hormonal fluctuations and ovulation cycle.


Natural Cycle IVF  is  in vitro fertilisation  (IVF) using either of the following procedures:


The first baby conceived through this method of IVF was  Louise Brown , the world's first 'test-tube baby.' However, as the field of infertility medicine progressed the protocol drifted away from this method and began incorporating the use of fertility drugs to promote greater production of eggs during one cycle. The idea behind this was to increase the number of eggs a woman can produce per cycle, thus increasing their chance of producing an embryo that will result in a live birth.  However, as this protocol to use more and more infertility drugs became the conventional IVF that we know today, many physicians began seeing the risks associated with conventional IVF.  Dr. Osamu Kato, the founder of the Kato Ladies Clinic, spearheaded the movement to begin a more natural and mild protocols for IVF. [ 4 ]


With no hyperstimulation drugs, the treatment cycle relies on the spontaneous development of one  follicle  only and therefore the aspiration of only one egg from the follicle (it is possible however that the cycle can have more than one egg or no eggs).
 GnRH antagonists  may still be given for  ovulation suppression . In addition the patient will need to take  hCG  (as with other less invasive treatments such as ovulation monitoring and  intrauterine insemination ) to time egg collection as well as progesterone  pessaries  to supplement the body’s  progesterone  levels. Progesterone aids implantation and supports  pregnancy  in its early stages.


It can be suitable for women who want to avoid ovarian stimulation or  fertility drugs  as a matter of choice, and for those for whom there may be no other choice, such as women at risk of hormone-related cancers. There is no suppression of the ovaries and associated  menopausal  symptoms and the treatment cycle is completed within a woman’s own  menstrual cycle .


There are no side-effects such as  ovarian hyperstimulation syndrome  (OHSS),  bloating , mood changes or other concerns relating to ovarian stimulation. Due to the effect of  ovarian stimulation  drugs on the body, patients undergoing stimulation cannot pursue consecutive cycles of treatment and need to take 2–3 months break between treatment cycles. In contrast, natural cycle patients can repeat their treatment in consecutive cycles. As only one  embryo  is transferred in a natural cycle, there is virtually no risk of a  multiple pregnancy . Furthermore, ovarian stimulation drugs are expensive and this means that the cost of each cycle is significantly less.


The success rate per cycle is low compared to stimulated  IVF .  HFEA  has estimated the  live birth rate  to be approximately 1.3% per IVF cycle using no hyperstimulation drugs for women aged between 40–42. [ 5 ]  There is also a small risk of spontaneous  ovulation  before egg collection. As a consequence, there is a need for a much larger number of cycles before achieving a live birth on average, in turn resulting in an average cost per live birth that is larger than with conventional IVF.


Natural cycle IVF is not suitable for those who do not ovulate spontaneously.


Mild IVF, [ 6 ]  sometimes called Soft IVF or IVF Lite, is aimed at producing 2-7 eggs. It does not involve shutting down the hormones for 2 weeks. It is conducted in the woman’s natural  menstrual cycle . Smaller dosages of stimulating drugs are given for a shorter period to help ripen the 2-7 eggs. Spontaneous  ovulation  is blocked with injections so that eggs could be collected. Some authors claim that it is safer, less expensive [ 7 ]  and avoids side-effects associated with suppression of  hormones  in a conventional IVF cycle. Others question the claim of economic superiority of mild IVF. One study, after comparing mild IVF and conventional IVF in good-prognosis patients, concluded that there was no cost difference between the two protocols on the basis of a "take home baby." [ 8 ]   Mild IVF reduces the risk of Ovarian Hyperstimulation Syndrome (OHSS).


Ovarian culture  is an  in-vitro  process that allows for the investigation of the development, toxicology and pathology of the  ovary . [ 1 ]  This technique can also be used to study possible applications of fertility treatments e.g. isolating oocytes from primordial ovarian follicles that could be used for fertilisation. [ 1 ]


There are several culture systems which can be employed to investigate ovarian and follicular growth and development. [ citation needed ]


The culture of intact ovaries supports the formation and development of primordial follicles. Ovaries are dissected from neonatal mouse pups and placed into ovarian culture medium containing  Bovine Serum Albumen  (BSA) dissolved in α-Minimal Essential Media (αMEM). The cultures are maintained in a 37°C, 5% CO 2  incubator and then the ovaries are frozen or fixed to facilitate further study. [ 1 ]


This method of culturing supports the growth of individual follicles from late  pre-antral  to pre-ovulatory stage. This system allows follicle growth and hormone production to be studied. The ovaries of young mice (19–23 days) are removed and halved, and follicles are identified under a microscope. Late pre-antral follicles are identified as having a diameter of 180-200 μm and containing 2-3 layers of  granulosa cells . Follicles are manually dissected and then examined for suitability to culture. Follicles are chosen for culture only if they are healthy (diameter of 190 ± 10 μm; translucent; without dark atretic areas; intact  basal lamina .) Wells containing follicle culture medium (α-Minimal Essential Media, recombinant human follicle stimulating hormone, ascorbic acid and adult female mouse serum) is overlaid with sterilised silicon oil, which prevents medium evaporation. A follicle is placed at the bottom of each well and maintained in a 37°C, 5% CO 2  incubator, being moved into a well containing fresh medium for up to 6 days. If growth measurements are being taken visually the distortion due to the oil layer must be accounted for. Follicles are frozen or fixed so further analysis can be performed. [ 1 ]


By culturing 2 follicles in close proximity, follicle-follicle interactions can be examined. The follicles may grow together to form a two-follicle unit. The follicles are dissected from the ovaries as above, then placed in contact with each other in pairs, in a well with follicle culture medium and sterilised silicon oil. Follicles from different genetic sources can be co-cultured so that tissue origins can be differentiated within the co-culture. The medium is replaced every 2 days and after 6 days the culture is fixed or frozen for further processing. [ 1 ]


This method allows follicle-ovary interactions to be studied.  The ovaries and follicles are dissected as above and then one follicle is placed in contact with one pole of a neonatal ovary on a plate. The follicle-ovary plate is cultured in follicle culture medium at 37 °C, 5% CO 2  for up to 5 days. At this point the co-culture is frozen or fixed before further processing. To facilitate differentiation between tissue origins the ovary and the follicle should be from different genetic sources. [ 1 ]


At present research within the field of  reproductive toxicology  is principally carried out  in vivo , however new culture methods have been developed with the aim of allowing ovarian follicles to be grown  in vitro . [ 3 ]  These new methods allow us to culture isolated ovarian follicles, embryos, ovaries (whole organ or only part of the tissue), and embryonic stem cells.  [ 1 ] Ovarian cultures are useful to research as they can allow us to replicate systematic follicle development, periodical ovulation, and follicle atresia in an environment with modulated culture conditions. [ 1 ] The ability of in vitro ovarian cultures to detect damage to the ovary and its specialised structures of the follicles and oocytes, allows for faster screening of potential developmental and/or reproductive toxicants. [ 3 ]  Therefore, ovarian culture systems have become increasingly widely used in reproductive biology and toxicology. [ 3 ]


Culture of the whole ovary or ovarian fragments allows evaluation of various parameters in a controlled way and, therefore, has the potential for more complete reproductive toxicity studies. [ 3 ]  A big advantage of ovarian culture is the ability to evaluate the effect of drugs on the pool of primordial follicles that make up the ovarian reserve. However, this strategy is restricted regarding the duration of culture time, as short periods may not be sufficient to ensure follicular development. [ 4 ]  On the contrary, cells may be negatively affected by longer periods of culture. [ 1 ]


Most in vitro toxicology studies use female mice and rat models. These species have been selected to assess the adverse effects of drugs on reproductive function and fertility, due to ease of handling and small size. [ 5 ]  Additionally, these species have been well characterised; anatomically, physiologically, and genetically. Their short life cycles make it convenient to assess gestation, breastfeeding, and puberty. [ 5 ]  The relevance of animal studies for toxicological risk assessment in heterogeneous human populations remains undetermined as it is unknown if the results obtained can be extrapolated to humans. [ 2 ]


The use of in vivo maturation in ovarian culture would eliminate the risk of  Ovarian Hyperstimulation Syndrome  during IVF in patients with  polycystic ovary syndrome  (PCOS).  [ 1 ] For those without PCOS, in vitro maturation still has advantages as the process is less intense as superovulation is not required. [ 6 ]  Principles of ovarian culture can be applied to women who are resistant to  FSH  or oestrogen sensitive tumours. [ 6 ]  In comparison to IVF, cells used in vitro maturation are harvested at a smaller size, immature and arrested at Metaphase I stage of meiosis. [ 6 ]  Once in the lab they undergo maturation to Metaphase II. [ 6 ]


Ovarian tissue can be harvested before ovarian damaging treatments and re-implanted at a later stage using cryopreservation. [ 5 ]  However, this method is associated with the recurrence of malignancy in those with ovarian cancer and leukaemia. [ 5 ]  In theory, ovarian tissue culture is a safer method to produce mature oocytes for fertilisation in these patients. [ 5 ]


Partner-assisted reproduction ,  reception of oocytes from partner  ( ROPA ),  reciprocal IVF,   shared motherhood ,  partner IVF  or  co-IVF  is a method of  family building  that is used by couples who both possess  female reproductive organs . The method uses  in vitro fertilization  (IVF), a method that means  eggs  are removed from the  ovaries , fertilized in a laboratory, and then one or more of the resulting  embryos  are placed in the  uterus  to hopefully create a  pregnancy . Reciprocal IVF differs from standard IVF in that two partners are involved: the eggs are taken from one partner, and the other partner carries the pregnancy. [ 1 ]  In this way, the process is mechanically identical to IVF with  egg donation . [ 2 ] [ 3 ]  Reciprocal IVF offers the highest chance for pregnancy and a lower chance of a multiple births. [ citation needed ]


This process was first introduced in  Spain  in 2007 at the CEFER institute. [ 3 ]


Reciprocal IVF is a process that involves steps both for the genetic mother and the gestational mother. Below lists the  one-by-one steps a couple undergoing this procedure have to go through.


The decision to choose which partner is going to be the egg donor and which partner is going to be the gestational carrier can be based on personal reasons or medical reasons. [ 12 ]  When focusing strictly on medical advantages, the egg donor tends to be the younger partner, the partner with the stronger ovarian reserve, or the partner without known genetic abnormalities.


The gestational carrier tends to be the partner who has previously given birth, is fit for pregnancy, or the partner with the healthiest uterus. [ 12 ]  These factors can be determined by a full fertility workup by a healthcare provider prior to starting the reciprocal IVF process. [ 12 ]


While some couples base their decision on medical reasons, personal reasons may also be a fundamental deciding factor. Many couples base their decision on emotional and/or psychological reasons of who wants to be the donor and who wants to carry the child to term. [ 12 ]


Identity reasons may also play a factor in a couple's decision. For instance, some partners who are biologically able may not want to be the gestational carrier because it misaligns with their gender identity or because testosterone regimens may need altering or cessation. [ 12 ]  However, while the research is limited, successful reciprocal IVF using oocytes from a transgender male who remained on testosterone throughout the entire process has been documented. [ 13 ]


The timeline of reciprocal IVF depends on if a couple chooses to undergo a fresh or a frozen transfer:


Epigenetics  includes the changes that occur in the  gene expression , but that does not alter the DNA sequence. These changes may be the result of external or environmental factors. There are multiple studies  [ 15 ]  related to epigenetics that show that the surrogate mother can also play an important role in the development of the embryo. This is because the prenatal environment of the uterus, where the embryo will stay for 9 months, and also the nutrients that reach it, have great relevance in the gene expression. Therefore, it is important that the birth mother have a healthy lifestyle and habits, in absence of toxics such as alcohol or tobacco, among others.


After  fertilisation , the  endometrial  fluid nourishes the  embryo  and regulates its development, before being replaced in this function by the  placenta  in the later stages of  pregnancy . This embryo-mother communication is finely regulated by numerous cellular pathways, including  epigenetic  mechanisms. Among the substances transported in the endometrial fluid,  extracellular vesicles  are one of the most important mediators, especially in relation to their ability to transport  ncRNAs . Primary human endometrial endothelial cells were found to internalize  miRNAs  into vesicles and actively secrete large amounts of  exosomes . These are internalized by embryonic  trophoblast  cells and may influence their epigenetic patterns. Even though the capability of numerous ncRNAs (both maternal and embryo-derived) of being transmitted through endometrial fluid has been clearly established, the role of such molecules in the regulation of pivotal phases of  implantation  and early embryonic development still requires much needed investigation. [ 16 ]


This phenomenon has important implications for patients who have to resort to using the ROPA method. Although the embryo does not carry the genetic information of the gestating mother, her  uterus  can influence the epigenetic characteristics of her future baby. This can help these women to overcome genetic grief, since they will be able to influence the  phenotype  of their child even if they do not share  DNA . [ 17 ]


Similar to reciprocal IVF, partners can choose concurrent IVF or double reciprocal IVF. This method is essentially two simultaneous reciprocal IVF processes, where both partners become pregnant at the same time using embryos from the opposite partner. [ 18 ]  This process allows both partners to undergo embryo transfers and simultaneous gestational carrying. There are potential considerations for concurrent IVF, including increased costs and potential stress from two childbirth deliveries. [ 18 ]


Partners also have the option to choose Effortless IVF. This method of reciprocal IVF uses an FDA-approved capsule (called INVOcell) to combine the donor sperm and egg, rather than reproduction in a lab. [ 18 ]  The partner who is acting as the egg donor will place the capsule back inside the vagina to incubate for around five days. Once the embryo begins development, it is removed and typically frozen. The partner who is acting as the gestational carrier will then prepare for embryo transfer and carry the resulting child to term. [ 18 ]


In most states, insurance coverage for IVF is not mandated and varies by state. Insurance providers do not typically cover the costs of reciprocal IVF unless it is medically necessary. [ 7 ]  Only 25% of Americans have coverage for IVF. [ 14 ]  On average, the cost of one IVF cycle costs about $12,000, which does not include fertility medications that typically range from $3,000 to $10,000. [ 7 ]  The average cost of reciprocal IVF in the United States is over $20,000 but varies based on the clinic and medication protocols. [ 14 ]


The cost of reciprocal IVF is more than IVF due to additional costs including donor sperm fees, legal fees, and fertility medications. [ 11 ]


As reciprocal IVF is a fairly new process first established in 2009, one study from 2017 found that 60% of couples undergoing IVF with an average donor age of 32 achieved pregnancy. [ 19 ]  A study published in February 2018 found a 60% live birth rate in a group of 120 lesbian couples who underwent reciprocal IVF. [ 20 ]  The subject of the research are European patients only.


The success rate of reciprocal IVF heavily depends on the age of the birth mother. For women under age 35, there is a 41-43% success rate. For women over 40, there is a 13-18% success rate. [ 21 ]


As IVF is not always successful on the first attempt, it is important that couples understand that multiple cycles may be required to achieve a pregnancy. [ 14 ]


For couples who choose to undergo reciprocal IVF, potential legal issues to be addressed may vary between jurisdictions. [ 5 ]  One potential issue is the necessity to make sure the partner who donates the egg is a legal parent of the child. [ 4 ]  This can include getting the name on the birth certificate at the time of birth or may require legally adopting the child. [ 5 ] [ 4 ]  These steps help establish equal parental rights for both the gestational carrier and the egg donor. [ 4 ]  A legal attorney who specializes in reproductive law can be a useful resource for navigating the specific legal aspects state by state. [ 4 ]


In 2023, the Hong Kong High Court ruled that a lesbian who provided an egg but did not carry the baby through reciprocal IVF was a “parent at common law” and that exclusion from parental status was discriminatory. [ 22 ] [ 23 ]


Preimplantation genetic diagnosis  ( PGD  or  PIGD ) is the  genetic  profiling of  embryos  prior to  implantation  (as a form of  embryo profiling ), [ 1 ]  and sometimes even of  oocytes  prior to  fertilization . PGD is considered in a similar fashion to  prenatal diagnosis . When used to screen for a specific  genetic disease , its main advantage is that it avoids selective  abortion , as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to  assisted reproductive technology , and requires  in vitro fertilization  (IVF) to obtain  oocytes  or embryos for evaluation. Embryos are generally obtained through  blastomere  or  blastocyst   biopsy . The latter technique has proved to be less deleterious for the embryo, therefore it is advisable to perform the biopsy around day 5 or 6 of development. [ 2 ]


The world's first PGD was performed by Handyside, [ 3 ]  Kontogianni and Winston at the Hammersmith Hospital in London. "Female embryos were selectively  transferred  in five couples at risk of  X-linked disease , resulting in two twin and one singleton  pregnancy ." [ 3 ] [ 4 ]


The term preimplantation genetic screening (PGS) refers to the set of techniques for testing whether embryos (obtained through IVF/  ICSI  have an abnormal number of  chromosomes  ( aneuploidy ). PGS is also called aneuploidy screening. PGS was renamed preimplantation genetic diagnosis for aneuploidy (PGD-A) by the Preimplantation Genetic Diagnosis International Society (PGDIS) in 2016. [ 5 ]


The PGD allows studying the DNA of eggs or embryos to select those that carry certain  mutations  for genetic diseases. It is useful when there are previous chromosomal or genetic disorders in the family and within the context of in vitro fertilization programs. [ 6 ]
These kinds of tests are needed because hereditary disorders are related with 20% of child deaths in developed countries. It is estimated that, as a whole, they are responsible for around 18% of pediatric hospital admissions.


The procedures may also be called "preimplantation genetic profiling" to adapt to the fact that they are sometimes used on oocytes or embryos prior to implantation for other reasons than diagnosis or screening. [ 7 ]


Procedures performed on  sex cells  before fertilization may instead be referred to as methods of  oocyte selection  or  sperm selection , although the methods and aims partly overlap with PGD.


In 1968,  Robert Edwards  and  Richard Gardner  reported the successful identification of the sex of rabbit  blastocysts . [ 8 ]  It was not until the 1980s that human IVF was fully developed, which coincided with the breakthrough of the highly sensitive  polymerase chain reaction  (PCR) technology. Handyside, Kontogianni and Winston's first successful tests happened in October 1989, with the first births in 1990 [ 9 ]  though the preliminary experiments had been published some years earlier. [ 10 ] [ 11 ]  In these first cases, PCR was used for sex determination of patients carrying  X-linked  diseases. A report in 2001 on worldwide use of PGD since 1990 reported that embryo or polar body biopsy [ 12 ]  had occurred in more than 3000 clinical cycles, with a 24% pregnancy rate, which was comparable with assisted reproductive practices which do not involve biopsy.


Elena Kontogianni was studying for her PhD at the Hammersmith Hospital, on single-cell PCR for sexing, which she did by amplifying a repeated region of the Y chromosome. [ 3 ]  It was this approach that she used for the world's first PGD cases. [ 4 ]


Female embryos were selectively  transferred  in five couples at risk of X-linked disease, resulting in two twins and one singleton pregnancy. Because the Y chromosome region Kontogianni was amplifying contained many repeats, it was more efficient than trying to amplify a unique region. A band on the PCR gel indicated that the embryo was male and the absence of a band indicated that the embryo was female. However, amplification failure or an anucleate blastomere also resulted in absence of a band on the PCR gel. To reduce the risk of misdiagnosis, Kontogianni went on to co-amplify sequences on the X and Y (Kontogianni et al., 1991). [ 13 ]  At that time nothing was known about allele dropout, cumulus cell contamination, or amplification failure from single cells. During the 1980s, human IVF embryos were exclusively transferred on day two of development as the culture medium used was incapable of reliably growing embryos past this stage. Since the  biopsy  was to be performed on day three, the first diagnoses were all performed in one day, with transfer of the embryos late on day three. A comparison of day two and day three transfers indicated that this would not adversely affect pregnancy rates. The worry of embryos arresting was so high that some transfers took place in the early hours of day four so that the embryos were removed from culture as soon as possible. There were many evenings at the Hammersmith when a transfer was performed at 1 a.m. on day four and researchers returned to the laboratory at 7 a.m. to start the next case. Winston helped deliver most of the first PGD babies.


PGD became increasingly popular during the 1990s when it was used to determine a handful of severe genetic disorders, such as  sickle-cell anemia ,  Tay–Sachs disease ,  Duchenne's muscular dystrophy , and  beta-thalassemia . [ 14 ]


PGD is used primarily for genetic disease prevention, by selecting only those embryos that do not have a known genetic disorder. PGD may also be used to increase chances of successful pregnancy, to match a sibling in  HLA type  in order to be a donor, to have less cancer predisposition, and for  sex selection . [ 2 ] [ 15 ] [ 16 ] [ 17 ]


PGD is frequently employed for the detection of autosomal dominant, autosomal recessive, and X-linked abnormalities. However, its utilization in screening for mitochondrial disorders is less common, primarily due to the unpredictable characteristics of mitochondrial heteroplasmy. In cases of mitochondrial heteroplasmy, some mitochondria within a cell bear the mutation, while others do not. The ratio of mutant mitochondria plays a crucial role in determining both the expression of the disease (its impact on offspring) and the severity of the disease. [ 18 ]


PGD is available for a large number of  monogenic disorders —that is, disorders due to a single gene only ( autosomal recessive ,  autosomal dominant  or  X-linked )—or of chromosomal structural aberrations (such as a balanced  translocation ). PGD helps these couples identify embryos carrying a genetic disease or a chromosome abnormality, thus avoiding diseased offspring. The most frequently diagnosed autosomal recessive disorders are  cystic fibrosis , Beta- thalassemia ,  sickle cell disease  and  spinal muscular atrophy  type 1. The most common dominant diseases are  myotonic dystrophy ,  Huntington's disease  and  Charcot–Marie–Tooth disease ; and in the case of the X-linked diseases, most of the cycles are performed for  fragile X syndrome ,  haemophilia A  and  Duchenne muscular dystrophy . Though it is quite infrequent, some centers report PGD for  mitochondrial  disorders or two indications simultaneously.


PGD is also now being performed in a disease called  hereditary multiple exostoses  (MHE/MO/HME).


In addition, there are infertile couples who carry an inherited condition and who opt for PGD as it can be easily combined with their IVF treatment.


Preimplantation genetic profiling (PGP) has been suggested as a method to determine  embryo quality  in  in vitro fertilization , in order to select an embryo that appears to have the greatest chances for successful pregnancy. However, as the results of PGP rely on the assessment of a single cell, PGP has inherent limitations as the tested cell may not be representative of the embryo because of  mosaicism . [ 19 ]  Furthermore, a study found that diagnoses of the biopsies from the same embryos at two separate laboratories matched up only 50% of the time. [ 20 ]


A systematic review and meta-analysis of existing  randomized controlled trials  came to the result that there is no evidence of a beneficial effect of PGP as measured by  live birth rate . [ 19 ]  On the contrary, for women of advanced maternal age, PGP significantly lowers the live birth rate. [ 19 ]  Technical drawbacks, such as the invasiveness of the biopsy, and chromosomal mosaicism are the major underlying factors for inefficacy of PGP. [ 19 ]  Normal live births of healthy offspring after transfers of embryos deemed aneuploid by PGP have been reported worldwide. [ 21 ]


Alternative methods to determine  embryo quality  for prediction of pregnancy rates include microscopy as well as profiling of  RNA  and  protein  expression.


Human leukocyte antigen  (HLA) typing of embryos, so that the child's HLA matches a sick sibling, availing for  cord-blood stem cell donation . [ 22 ] [ 23 ]  The child is in this sense a " savior sibling " for the recipient child. HLA typing has meanwhile become an important PGD indication in those countries where the law permits it. [ 24 ]  The HLA matching can be combined with the diagnosis for monogenic diseases such as  Fanconi anaemia  or  beta thalassemia  in those cases where the ailing sibling is affected with this disease, or it may be exceptionally performed on its own for cases such as children with  leukaemia . The main ethical argument against is the possible exploitation of the child, although some authors maintain that the  Kantian imperative  is not breached since the future donor child will not only be a donor but also a loved individual within the family. [ citation needed ]


A more recent application of PGD is to diagnose late-onset diseases and (cancer) predisposition syndromes. Since affected individuals remain healthy until the onset of the disease, frequently in the fourth decade of life, there is debate on whether or not PGD is appropriate in these cases. Considerations include the high probability of developing the disorders and the potential for cures. For example, in predisposition syndromes, such as  BRCA mutations  which predispose the individual to breast cancer, the outcomes are unclear. Although PGD is often regarded as an early form of prenatal diagnosis, the nature of the requests for PGD often differs from those of prenatal diagnosis requests made when the mother is already pregnant. Some of the widely accepted indications for PGD would not be acceptable for prenatal diagnosis. [ citation needed ]


A woman carrying the gene for early-onset Alzheimer's disease used preimplantation genetic diagnosis (PGD) to ensure that her child would not inherit this condition. Ethical questions arise regarding the decision to allow an individual aware of their own susceptibility to a late-onset disease to have a child free of the gene, despite the risk that the child may lose a parent prematurely. Some argue that this decision is ethical, asserting that the desire for reproduction is as legitimate for these individuals as for others seeking infertility services. Comparisons are drawn with other medical situations, such as assisted reproduction for individuals with HIV or other serious illnesses. Although the child may face risks of early bereavement, the argument put forth is that the psychological trauma does not render the child's life devoid of clear benefits. Therefore, assisting parents in reproducing in these circumstances is not considered to cause undue or unnecessary suffering to the child. [ 25 ] [ 26 ]


Preimplantation genetic diagnosis provides a method of  prenatal sex discernment  even before implantation, and may therefore be termed  preimplantation sex discernment . Potential applications of preimplantation sex discernment include:


In the case of families at risk for  X-linked diseases , patients are provided with a single PGD assay of gender identification.  Gender selection  offers a solution to individuals with X-linked diseases who are in the process of getting pregnant. The selection of a female embryo offspring is used in order to prevent the transmission of X-linked Mendelian recessive diseases. Such X-linked Mendelian diseases include  Duchenne muscular dystrophy  (DMD), and hemophilia A and B, which are rarely seen in females because the offspring is unlikely to inherit two copies of the recessive allele. Since two copies of the mutant X allele are required for the disease to be passed on to the female offspring, females will at worst be carriers for the disease but may not necessarily have a dominant gene for the disease. Males on the other hand only require one copy of the mutant X allele for the disease to occur in one's phenotype and therefore, the male offspring of a carrier mother has a 50% chance of having the disease. Reasons may include the rarity of the condition or because affected males are reproductively disadvantaged. Therefore, medical uses of PGD for selection of a female offspring to prevent the transmission of X-linked Mendelian recessive disorders are often applied. Preimplantation genetic diagnosis applied for gender selection can be used for non-Mendelian disorders that are significantly more prevalent in one sex. Three assessments are made prior to the initiation of the PGD process for the prevention of these inherited disorders. In order to validate the use of PGD, gender selection is based on the seriousness of the inherited condition, the risk ratio in either sex, or the options for disease treatment. [ citation needed ]


PGD has occasionally been used to select an embryo for the presence of a particular disease or disability, such as deafness, in order that the child would share that characteristic with the parents. [ 29 ]


The potential controversial use of PGD could arise with genetic tests targeting non-medical traits such as hearing, sexual orientation, height, beauty, or intelligence. Some tests, like those for GJB2 mutations linked to hereditary deafness, might lead to requests for PGD to avoid or favor these traits. Ethical concerns include potential harm to affected communities, such as the deaf. Similar questions would arise with a genetic test for sexual orientation, raising concerns about discrimination. Some insist on a complete ban on selecting such traits, fearing more serious implications like genetic engineering of offspring. However, definitive judgments on these issues cannot be made until such tests are closer to practical reality. [ 30 ] [ 31 ] [ 32 ] [ 33 ] [ 34 ] [ 35 ] [ 36 ]


There are three distinguished types of Preimplantation Genetic Testing (PGT) depending on the defects evaluated.


PGT-A , also called preimplantational genetic screening (PGS), improves pregnancy rates by allowing the discard of  aneuploids  and the selection of euploid embryos for transfer. Euploid embryos are more likely to implant and develop into a healthy pregnancy.  NGS  and  FISH  are the most frequently techniques used for the diagnosis of monosomies, trisomies and poliploidies. Primary candidates for PGT-A can include the following:


PGT-A is particularly beneficial for women of advanced maternal age. As women age, especially beyond 35 years, the quality of their eggs declines due to an increased risk of errors during the meiotic division of oocytes. These errors lead to chromosomal abnormalities, such as aneuploidies (monosomies, trisomies, and polyploidies), which can impair embryo development and result in failed pregnancies or genetic disorders.


Because of this, PGT-A has become an essential tool for identifying and selecting euploid embryos (embryos with the correct number of chromosomes) for transfer, significantly improving the chances of a successful pregnancy. Studies have shown that the probability of chromosomal abnormalities increases with maternal age, making PGT-A an important step in fertility treatments like in vitro fertilization (IVF) for older women.


PGT-M  evaluates  monogenic diseases  are being evaluated in the embryo. Monogenic disorders are caused by single-gene mutations (autosomal recessive, autosomal dominant,  X-linked ). Previously,  PCR  was used for PGT-M. Recently, array and NGS technology are used.


PGT-SR  takes every structural abnormality in the chromosome into account ( translocations ,  inversions ,  duplications ,  insertions ,  deletions ). Techniques used include PCR, FISH and NGS.


PGD is a form of genetic diagnosis performed prior to implantation. This implies that the patient's oocytes should be fertilized  in vitro  and the embryos kept in culture until the diagnosis is established. It is also necessary to perform a biopsy on these embryos in order to obtain material on which to perform the diagnosis. The diagnosis itself can be carried out using several techniques, depending on the nature of the studied condition. Generally, PCR-based methods are used for monogenic disorders and FISH for chromosomal abnormalities and for sexing those cases in which no PCR protocol is available for an X-linked disease. These techniques need to be adapted to be performed on blastomeres and need to be thoroughly tested on single-cell models prior to clinical use. Finally, after embryo replacement, surplus good quality unaffected embryos can be cryopreserved, to be thawed and transferred back in a next cycle.


Currently, all PGD embryos are obtained by  assisted reproductive technology , although the use of natural cycles and  in vivo  fertilization followed by uterine lavage was attempted in the past and is now largely abandoned. In order to obtain a large group of oocytes, the patients undergo controlled ovarian stimulation (COH). COH is carried out either in an agonist protocol, using gonadotrophin-releasing hormone (GnRH) analogues for pituitary desensitisation, combined with human menopausal gonadotrophins (hMG) or recombinant follicle-stimulating hormone (FSH), or an antagonist protocol using recombinant FSH combined with a GnRH antagonist according to clinical assessment of the patient's profile (age, body mass index (BMI), endocrine parameters). hCG is administered when at least three follicles of more than 17 mm [ verification needed ]  mean diameter are seen at transvaginal ultrasound scan. Transvaginal ultrasound-guided oocyte retrieval is scheduled 36 hours after hCG administration. Luteal phase supplementation consists of daily intravaginal administration of 600 μg of natural micronized progesterone.


Oocytes are carefully denudated from the cumulus cells, as these cells can be a source of contamination during the PGD if PCR-based technology is used. In the majority of the reported cycles,  intracytoplasmic sperm injection  (ICSI) is used instead of IVF. The main reasons are to prevent contamination with residual sperm adhered to the zona pellucida and to avoid unexpected fertilization failure. The ICSI procedure is carried out on mature metaphase-II oocytes and fertilization is assessed 16–18 hours after. The embryo development is further evaluated every day prior to biopsy and until transfer to the woman's uterus. During the cleavage stage, embryo evaluation is performed daily on the basis of the number, size, cell-shape and fragmentation rate of the blastomeres. On day 4, embryos were scored in function of their degree of compaction and blastocysts were evaluated according to the quality of the throphectoderm and inner cell mass, and their degree of expansion.


As PGD can be performed on cells from different developmental stages, the biopsy procedures vary accordingly. Theoretically, the biopsy can be performed at all preimplantation stages, but only three have been suggested: on unfertilised and fertilised oocytes (for polar bodies, PBs), on day three cleavage-stage embryos (for blastomeres) and on blastocysts (for trophectoderm cells).


The biopsy procedure always involves two steps: the opening of the  zona pellucida  and the removal of the cells. There are different approaches to both steps, including mechanical, chemical, and physical ( Tyrode's acidic solution ) and laser technology for the breaching of the zona pellucida, extrusion or aspiration for the removal of PBs and blastomeres, and herniation of the trophectoderm cells.


A  polar body biopsy  is the  sampling  of a  polar body , which is a small  haploid  cell that is formed concomitantly as an  egg cell  during  oogenesis , but which generally does not have the ability to be  fertilized . Compared to a  blastocyst biopsy , a polar body biopsy can potentially be of lower costs, less harmful side-effects, and more  sensitive  in detecting abnormalities. [ 38 ]  The main advantage of the use of polar bodies in PGD is that they are not necessary for successful fertilisation or normal embryonic development, thus ensuring no deleterious effect for the embryo. One of the disadvantages of PB biopsy is that it only provides information about the maternal contribution to the embryo, which is why cases of maternally inherited autosomal dominant and X-linked disorders that are exclusively maternally transmitted can be diagnosed, and autosomal recessive disorders can only partially be diagnosed. Another drawback is the increased risk of diagnostic error, for instance due to the degradation of the genetic material or events of recombination that lead to heterozygous first polar bodies.


Cleavage-stage biopsy is generally performed the morning of day three post-fertilization, when normally developing embryos reach the eight-cell stage. The biopsy is usually performed on embryos with less than 50% of anucleated fragments and at an eight-cell or later stage of development. A hole is made in the zona pellucida and one or two  blastomeres  containing a nucleus are gently aspirated or extruded through the opening.
The main advantage of cleavage-stage biopsy over PB analysis is that the genetic input of both parents can be studied. On the other hand, cleavage-stage embryos are found to have a high rate of  chromosomal mosaicism , putting into question whether the results obtained on one or two blastomeres will be representative for the rest of the embryo. It is for this reason that some programs utilize a combination of PB biopsy and blastomere biopsy. Furthermore, cleavage-stage biopsy, as in the case of PB biopsy, yields a very limited amount of tissue for diagnosis, necessitating the development of single-cell  PCR  and  FISH  techniques.
Although theoretically PB biopsy and blastocyst biopsy are less harmful than cleavage-stage biopsy, this is still the prevalent method. It is used in approximately 94% of the PGD cycles reported to the ESHRE PGD Consortium. The main reasons are that it allows for a safer and more complete diagnosis than PB biopsy and still leaves enough time to finish the diagnosis before the embryos must be replaced in the patient's uterus, unlike blastocyst biopsy.
Of all cleavage-stages, it is generally agreed that the optimal moment for biopsy is at the eight-cell stage. It is diagnostically safer than the PB biopsy and, unlike blastocyst biopsy, it allows for the diagnosis of the embryos before day 5. In this stage, the cells are still totipotent and the embryos are not yet compacting. Although it has been shown that up to a quarter of a human embryo can be removed without disrupting its development, it still remains to be studied whether the biopsy of one or two cells correlates with the ability of the embryo to further develop, implant, and grow into a full-term pregnancy.


Not all methods of opening the  zona pellucida  have the same success rate because the well-being of the embryo and blastomere may be impacted by the procedure used for the biopsy. Zona drilling with acid Tyrode's solution (ZD) was looked at in comparison to partial zona dissection (PZD) to determine which technique would lead to more successful pregnancies and have less of an effect on the embryo and/or blastomere. ZD uses a digestive enzyme like pronase which makes it a chemical drilling method. The chemicals used in ZD may have a damaging effect on the embryo. PZD uses a glass microneedle to cut the zona pellucida which makes it a mechanical dissection method that typically needs skilled hands to perform the procedure. In a study that included 71 couples, ZD was performed in 26 cycles from 19 couples and PZD was performed in 59 cycles from 52 couples. In the single-cell analysis, there was a success rate of 87.5% in the PZD group and 85.4% in the ZD group. The maternal age, number of oocytes retrieved, fertilization rate, and other variables did not differ between the ZD and PZD groups. It was found that PZD led to a significantly higher rate of pregnancy (40.7% vs 15.4%), ongoing pregnancy (35.6% vs 11.5%), and implantation (18.1% vs 5.7%) than ZD. This suggests that using the mechanical method of PZD in blastomere biopsies for preimplantation genetic diagnosis may be more proficient than using the chemical method of ZD. The success of PZD over ZD could be attributed to the chemical agent in ZD having a harmful effect on the embryo and/or blastomere. Currently, zona drilling using a laser is the predominant method of opening the zona pellucida. Using a laser is an easier technique than using mechanical or chemical means. However, laser drilling could be harmful to the embryo and it is very expensive for in vitro fertilization laboratories to use especially when PGD is not a prevalent process as of modern times. PZD could be a viable alternative to these issues. [ 39 ]


In an attempt to overcome the difficulties related to single-cell techniques, it has been suggested to biopsy embryos at the blastocyst stage, providing a larger amount of starting material for diagnosis. It has been shown that if more than two cells are present in the same sample tube, the main technical problems of single-cell PCR or FISH would virtually disappear. On the other hand, as in the case of cleavage-stage biopsy, the chromosomal differences between the inner cell mass and the  trophectoderm  (TE) can reduce the accuracy of diagnosis, although this  mosaicism  has been reported to be lower than in cleavage-stage embryos.


TE biopsy has been shown to be successful in animal models such as rabbits, [ 40 ]  mice [ 41 ]  and primates. [ 42 ]  These studies show that the removal of some TE cells is not detrimental to the further  in vivo  development of the embryo.


Human blastocyst-stage biopsy for PGD is performed by making a hole in the ZP on day three of  in vitro  culture. This allows the developing TE to protrude after blastulation, facilitating the biopsy. On day five post-fertilization, approximately five cells are excised from the TE using a glass needle or laser energy, leaving the embryo largely intact and without loss of inner cell mass. After diagnosis, the embryos can be replaced during the same cycle, or  cryopreserved  and transferred in a subsequent cycle.


There are two drawbacks to this approach, due to the stage at which it is performed. First, only approximately half of the preimplantation embryos reach the blastocyst stage. This can restrict the number of blastocysts available for biopsy, limiting in some cases the success of the PGD. McArthur and coworkers [ 43 ]  report that 21% of the started PGD cycles had no embryo suitable for TE biopsy. This figure is approximately four times higher than the average presented by the ESHRE PGD consortium data, where PB and cleavage-stage biopsy are the predominant reported methods. On the other hand, delaying the biopsy to this late stage of development limits the time to perform the genetic diagnosis, making it difficult to redo a second round of PCR or to rehybridize FISH probes before the embryos should be transferred back to the patient.


Sampling of  cumulus cells  can be performed in addition to a sampling of polar bodies or cells from the embryo. Because of the molecular interactions between cumulus cells and the oocyte,  gene expression profiling of cumulus cells  can be performed to estimate oocyte quality and the efficiency of an  ovarian hyperstimulation  protocol, and may indirectly predict  aneuploidy , embryo development and pregnancy outcomes. [ 44 ]


Traditional embryo biopsy can be invasive and costly. Therefore, researchers have an ongoing quest to find a less invasive methods for preimplantation  genetic testing . Studies on new non-invasive preimplantation genetics screening methods such as  blastocoel  fluid and spent embryo media have recently been published as an alternative to traditional methods. [ 45 ]


During a normal IVF process, good practice to vitrify embryos increases the chance of a healthy pregnancy. During the process of vitrification a developed blast is dehydrated and it and its blastocoel cavity collapses for the freezing process. There are many methods that have been used to facilitate the collapse including laser-pulse, repeated  micropipetting , microneedle puncture or microsuction. [ 46 ]  Normally this fluid would then be discarded, however with preimplantation genetic testing of BL, this fluid is saved and then tested for DNA. This DNA is thought to be from cells that have gone through  apoptosis  found in the developing embryo. [ 45 ]


Another method for less invasive preimplantation genetic testing involves testing the culture media the embryo has developed in. It has been noted that the embryo releases DNA fragments from the cells that have died within the incubation period. With this knowledge, scientists have reasoned that they could isolate this DNA and use it for preimplantation genetic testing. [ 45 ]


While there is conflicting evidence as to whether or not the more traditional methods of preimplantation genetic testing are harmful to the embryo, there are newer methods for less invasive and equally effective testing methods using blastocoel fluid and spent embryo media. Two problems with these alternatives are the minimal amount of DNA there is to work with and whether or not this technology is accurate. Both of these concerns were recently addressed by Kuznyetsov, who decided to use both methods, combining the amount of DNA retrieved from both techniques. Once the DNA was isolated it was used for preimplantation genetic testing. The results showed that when both methods (blastocyst fluid and embryo spent media) were used in combination, they showed a cordance rate for the whole chromosome copy of 87.5% when compared to the trophectoderm, 96.4% when compared to the whole blastocyst (gold standard). Additionally after amplification using this new method they were able to produce 25.0–54.0 ng/ul of DNA per sample. With traditional methods such as trophectoderm they collected 10 to 44 ng/ul. [ 45 ]


Fluorescent in situ hybridization  (FISH) and  polymerase chain reaction  (PCR) are the two commonly used, first-generation technologies in PGD. PCR is generally used to diagnose monogenic disorders and FISH is used for the detection of chromosomal abnormalities (for instance,  aneuploidy  screening or chromosomal translocations). Over the past few years, various advancements in PGD testing have allowed for an improvement in the comprehensiveness and accuracy of results available depending on the technology used. [ 47 ] [ 48 ]  Recently a method was developed allowing to fix metaphase plates from single blastomeres. This technique in conjunction with FISH, m-FISH can produce more reliable results, since analysis is done on whole metaphase plates [ 49 ]


In addition to FISH and PCR,  single cell genome sequencing  is being tested as a method of preimplantation genetic diagnosis. [ 50 ]  This characterizes the complete  DNA  sequence of the  genome  of the embryo.


FISH  is the most commonly applied method to determine the chromosomal constitution of an embryo. In contrast to  karyotyping , it can be used on interphase chromosomes, so that it can be used on PBs, blastomeres and TE samples. The cells are fixated on glass microscope slides and hybridised with DNA probes. Each of these probes are specific for part of a chromosome, and are labelled with a fluorochrome.


Dual FISH was considered to be an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction (PCR). [ 51 ]


Currently, a large panel of probes are available for different segments of all chromosomes, but the limited number of different  fluorochromes  confines the number of signals that can be analysed simultaneously.


The type and number of probes that are used on a sample depends on the indication. For sex determination (used for instance when a PCR protocol for a given X-linked disorder is not available), probes for the X and Y chromosomes are applied along with probes for one or more of the  autosomes  as an internal FISH control. More probes can be added to check for  aneuploidies , particularly those that could give rise to a viable pregnancy (such as a  trisomy 21 ). The use of probes for chromosomes X, Y, 13, 14, 15, 16, 18, 21 and 22 has the potential of detecting 70% of the aneuploidies found in spontaneous abortions.


In order to be able to analyse more chromosomes on the same sample, up to three consecutive rounds of FISH can be carried out. In the case of chromosome rearrangements, specific combinations of probes have to be chosen that flank the region of interest. The FISH technique is considered to have an error rate of 5–10%.


The main problem of the use of FISH to study the chromosomal constitution of embryos is the elevated mosaicism rate observed at the human preimplantation stage. A meta-analysis of more than 800 embryos came to the result that approximately 75% of preimplantation embryos are mosaic, of which approximately 60% are diploid–aneuploid mosaic and approximately 15% aneuploid mosaic. [ 52 ]  Li and co-workers [ 53 ]  found that 40% of the embryos diagnosed as aneuploid on day 3 turned out to have a  euploid  inner cell mass at day 6. Staessen and collaborators found that 17.5% of the embryos diagnosed as abnormal during PGS, and subjected to post-PGD reanalysis, were found to also contain normal cells, and 8.4% were found grossly normal. [ 54 ]  As a consequence, it has been questioned whether the one or two cells studied from an embryo are actually representative of the complete embryo, and whether viable embryos are not being discarded due to the limitations of the technique. Nevertheless, mosaic embryos can be transferred but only if there are not euploids available, having previously informed patients about the risks and doing a prenatal diagnose preferably.


Kary Mullis  conceived  PCR  in 1985 as an  in vitro  simplified reproduction of the  in vivo  process of  DNA replication . Taking advantage of the chemical properties of DNA and the availability of thermostable  DNA polymerases , PCR allows for the enrichment of a DNA sample for a certain sequence. PCR provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. It is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including PGD. Currently, many different variations exist on the PCR itself, as well as on the different methods for the posterior analysis of the PCR products.


When using PCR in PGD, one is faced with a problem that is nonexistent in routine genetic analysis: the minute amounts of available genomic DNA. As PGD is performed on single cells, PCR has to be adapted and pushed to its physical limits, and use the minimum amount of template possible: which is one strand. This implies a long process of fine-tuning of the PCR conditions and a susceptibility to all the problems of conventional PCR, but several degrees intensified. The high number of needed PCR cycles and the limited amount of template makes single-cell PCR very sensitive to contamination. Another problem specific to single-cell PCR is the allele drop out (ADO) phenomenon. It consists of the random non-amplification of one of the  alleles  present in a  heterozygous  sample. ADO seriously compromises the reliability of PGD as a heterozygous embryo could be diagnosed as affected or unaffected depending on which allele would fail to amplify. This is particularly concerning in PGD for  autosomal dominant  disorders, where ADO of the affected allele could lead to the transfer of an affected embryo.


Several PCR-based assays have been developed for various diseases like the triplet repeat genes associated with myotonic dystrophy and fragile X in single human somatic cells, gametes and embryos. [ 55 ]


The core philosophy of massive parallel sequencing used in NGS is adapted from shotgun sequencing developed to sequence longer sections of DNA. NGS technologies read the target DNA templates randomly. The target DNA or entire genome is broken into small pieces and then those DNA pieces are ligated to designated adapters for random reading during [ 56 ]  in-parallel DNA synthesis. The  read length  corresponds to the actual number of continuous sequenced bases. The read lengths are much shorter than with Sanger sequencing, which is why NGS results are called  short reads .


From 2014,  next-generation sequencing  (NGS) is being performed in the PGT. [ 57 ]  NGS is a group of techniques capable of sequencing great amounts of DNA at a reasonable cost and time. It can give us a general perspective of the complete embryo genome, including the  mitochondrial one . Those techniques are based on sequencing short reads around 400 bases each and overlapping these reads with powerful alignment software.


Likewise, NGS is used to detect aneuploidies in the 24 chromosomes and single-gene defects when there is an indication from the carrier parents. The main advantage is that NGS can combine the detection of both aneuploidies and monogenic diseases with a single biopsy and has reduced affordable costs, making it more accessible.


Two examples of NGS are the  pyrosequencing  and the  reversible dye terminator .


Pyrosequencing technique is based on sequencing by-synthesis principle and on the detection of released pyrophosphate during DNA synthesis. It employs a series of four enzymes to accurately detect nucleic acid sequences during the synthesis. [ 58 ]


Cycles of four  deoxynucleotide triphosphates  (dNTPs) are separately added to the reaction mixture iteratively. The cascade starts with a nucleic acid polymerization reaction in which inorganic  pyrophosphate  is released as a result of nucleotide incorporation by polymerase. Each nucleotide incorporation event is followed by release of inorganic pyrophosphate in a quantity equimolar to the amount of incorporated nucleotide. The released pyrophosphate is quantitatively converted to ATP by  ATP sulfurylase  in the presence of APS. The generated ATP drives the  luciferase -mediated conversion of  luciferin  to oxyluciferin, producing visible light in amounts that are proportional to the amount of ATPs. During this synthesis process, the DNA strand is extended by complementary nucleotides, and the DNA sequence is demonstrated by the pyrogram on a screen. The overall reaction from polymerization to light detection takes place within 3–4 seconds at room temperature. ATP sulfurylase converts pyrophosphate to ATP in approximately 1.5 seconds and the generation of light by luciferase takes place in less than 0.2 seconds.


Along with the benefits offered by these technologies, there are a number of challenges, both technical and ethical that must be addressed and solved before NGS technologies enter the clinical arena of embryo diagnosis. One limitation will be the interpretation of the massive sequence data generated by NGS technologies. Background polymorphisms must be distinguished from potentially disease-causing mutations and copy number variations. By selective recovery and subsequent sequencing of genomic loci of interest, generated data and efforts for analysis can be reduced significantly compared with a whole-genome sequencing approach. [ 56 ]


The establishment of a diagnosis in PGD is not always straightforward. The criteria used for choosing the embryos to be replaced after FISH or PCR results are not equal in all centres.
In the case of FISH, in some centres only embryos are replaced that are found to be chromosomally normal (that is, showing two signals for the gonosomes and the analysed autosomes) after the analysis of one or two blastomeres, and when two blastomeres are analysed, the results should be concordant. Other centres argue that embryos diagnosed as monosomic could be transferred, because the false monosomy (i.e. loss of one FISH signal in a normal diploid cell) is the most frequently occurring misdiagnosis. In these cases, there is no risk for an aneuploid pregnancy, and normal diploid embryos are not lost for transfer because of a FISH error. Moreover, it has been shown that embryos diagnosed as monosomic on day 3 (except for chromosomes X and 21), never develop to blastocyst, which correlates with the fact that these monosomies are never observed in ongoing pregnancies.


Diagnosis and misdiagnosis in PGD using PCR have been mathematically modelled in the work of Navidi and Arnheim and of Lewis and collaborators. [ 59 ] [ 60 ]  The most important conclusion of these publications is that for the efficient and accurate diagnosis of an embryo, two genotypes are required. This can be based on a linked marker and disease genotypes from a single cell or on marker/disease genotypes of two cells. An interesting aspect explored in these papers is the detailed study of all possible combinations of alleles that may appear in the PCR results for a particular embryo. The authors indicate that some of the genotypes that can be obtained during diagnosis may not be concordant with the expected pattern of linked marker genotypes, but are still providing sufficient confidence about the unaffected genotype of the embryo. Although these models are reassuring, they are based on a theoretical model, and generally the diagnosis is established on a more conservative basis, aiming to avoid the possibility of misdiagnosis. When unexpected alleles appear during the analysis of a cell, depending on the genotype observed, it is considered that either an abnormal cell has been analysed or that contamination has occurred, and that no diagnosis can be established. A case in which the abnormality of the analysed cell can be clearly identified is when, using a  multiplex PCR  for linked markers, only the alleles of one of the parents are found in the sample. In this case, the cell can be considered as carrying a monosomy for the chromosome on which the markers are located, or, possibly, as haploid. The appearance of a single allele that indicates an affected genotype is considered sufficient to diagnose the embryo as affected, and embryos that have been diagnosed with a complete unaffected genotype are preferred for replacement. Although this policy may lead to a lower number of unaffected embryos suitable for transfer, it is considered preferable to the possibility of a misdiagnosis.


Preimplantation genetic haplotyping  (PGH) is a PGD technique wherein a  haplotype  of  genetic markers  that have statistical associations to a target disease are identified rather than the  mutation  causing the disease. [ 61 ]


Once a panel of associated genetic markers have been established for a particular disease it can be used for all carriers of that disease. [ 61 ]  In contrast, since even a monogenic disease can be caused by many different mutations within the affected gene, conventional PGD methods based on finding a specific mutation would require mutation-specific tests. Thus, PGH widens the availability of PGD to cases where mutation-specific tests are unavailable.


PGH also has an advantage over FISH in that FISH is not usually able to make the differentiation between embryos that possess the balanced form of a  chromosomal translocation  and those carrying the homologous normal chromosomes. This inability can be seriously harmful to the diagnosis made. PGH can make the distinction that FISH often cannot. PGH does this by using polymorphic markers that are better suited at recognizing translocations. These polymorphic markers are able to distinguish between embryos that carried normal, balanced, and unbalanced translocations. FISH also requires more cell fixation for analysis whereas PGH requires only transfer of cells into polymerase chain reaction tubes. The cell transfer is a simpler method and leaves less room for analysis failure. [ 62 ]


Embryo transfer  is usually performed on day three or day five post-fertilization, the timing depending on the techniques used for PGD and the standard procedures of the  IVF  centre where it is performed.


With the introduction in Europe of the single-embryo transfer policy, which aims at the reduction of the incidence of multiple pregnancies after ART, usually one embryo or early blastocyst is replaced in the uterus. Serum hCG is determined at day 12. If a pregnancy is established, an ultrasound examination at 7 weeks is performed to confirm the presence of a fetal heartbeat. Couples are generally advised to undergo  PND  because of the, albeit low, risk of misdiagnosis.


It is not unusual that after the PGD, there are more embryos suitable for transferring back to the woman than necessary. For the couples undergoing PGD, those embryos are very valuable, as the couple's current cycle may not lead to an ongoing pregnancy.  Embryo cryopreservation  and later thawing and replacement can give them a second chance to pregnancy without having to redo the cumbersome and expensive ART and PGD procedures.


PGD/PGS is an invasive procedure that requires a serious consideration, according to Michael Tucker, Ph.D., Scientific Director and Chief Embryologist at Georgia Reproductive Specialists in Atlanta. [ 63 ]   One of the risks of PGD includes damage to the embryo during the biopsy procedure (which in turn destroys the embryo as a whole), according to Serena H. Chen, M.D., a New Jersey reproductive endocrinologist with IRMS Reproductive Medicine at Saint Barnabas. [ 63 ]   Another risk is cryopreservation where the embryo is stored in a frozen state and thawed later for the procedure. About 20% of the thawed embryos do not survive. [ 64 ] [ 65 ]   There has been a study indicating a biopsied embryo has a less rate of surviving cryopreservation. [ 66 ]   Another study suggests that PGS with cleavage-stage biopsy results in a significantly lower live birth rate for women of advanced maternal age. [ 19 ]   Also, another study recommends the caution and a long term follow-up as PGD/PGS increases the perinatal death rate in multiple pregnancies. [ 67 ]


In a mouse model study, PGD has been attributed to various long-term risks, including a weight gain and memory decline; a proteomic analysis of adult mouse brains showed significant differences between the biopsied and the control groups, of which many are closely associated with neurodegenerative disorders like Alzheimer's and Down syndrome. [ 68 ] [ 69 ]


PGD has raised ethical issues, although this approach could reduce reliance on fetal deselection during pregnancy.  The technique can be used for  prenatal sex discernment  of the embryo, and thus potentially can be used to select embryos of one sex in preference of the other in the context of " family balancing ". [ 12 ]  The use of PGD for gender variety has been reported in India, where an IVF programme in Bombay is now providing PGD to select male offspring as the second child of couples who have already had a daughter. Because of the importance of a male heir in India, those couples might have resorted to abortion if pregnant with a female fetus (even though illegal for that purpose). In that setting PGD for sex selection for gender variety appears to be justified. It may be possible to make other "social selection" choices in the future that introduce socio-economic concerns.  Only unaffected embryos are implanted in a woman's uterus; those that are affected are either discarded or donated to science. [ 70 ]


Objections to PGD based on its effect on embryos replay debates over abortion and embryo status that have occurred in many other contexts, from abortion to embryonic stem cell research. People who think that the embryo or fetus is a person will object to creating and destroying embryos, and oppose most uses of PGD. Others believe that preimplantation embryos are too rudimentary in development to have interests or rights, but that they deserve special respect as the first stage toward a new person. [ 12 ]


PGD has the potential to screen for genetic issues unrelated to  medical necessity , such as intelligence and beauty, and against negative traits such as disabilities. The medical community has regarded this as a counterintuitive and controversial suggestion. [ 71 ]  The prospect of a " designer baby " is closely related to the PGD technique, creating a fear that increasing frequency of genetic screening will move toward a modern  eugenics  movement. [ 12 ]  On the other hand, a principle of  procreative beneficence  is proposed, which is a putative  moral obligation  of  parents  in a position to select their children to favor those expected to have the best life. [ 72 ]  An argument in favor of this principle is that traits (such as empathy, memory, etc.) are "all-purpose means" in the sense of being of  instrumental value  in realizing whatever life plans the child may come to have. [ 73 ]  Walter Veit has argued that there is no intrinsic moral difference between 'creating' and 'choosing' a life, thus making eugenics a natural consequence of accepting the principle of procreative beneficence. [ 74 ]


In 2006, three percent of PGD clinics in the US reported having selected an embryo for the presence of a disability. [ 75 ]  Couples involved were accused of purposely harming a child. This practice is notable in dwarfism, where parents intentionally create a child who is a dwarf. [ 75 ]  In the selection of a  saviour sibling  to provide a matching bone marrow transplant for an already existing affected child, there are issues including the  commodification  and welfare of the donor child. [ 76 ]


By relying on the result of one cell from the multi-cell embryo, PGD operates under the assumption that this cell is representative of the remainder of the embryo. This may not be the case as the incidence of mosaicism is often relatively high. [ 77 ]  On occasion, PGD may result in a  false negative  result leading to the acceptance of an abnormal embryo, or in a  false positive  result leading to the deselection of a normal embryo.


Another problematic case is the cases of desired non-disclosure of PGD results for some genetic disorders that may not yet be apparent in a parent, such as  Huntington disease . It is applied when patients do not wish to know their carrier status but want to ensure that they have offspring free of the disease. This procedure can place practitioners in questionable ethical situations, e.g. when no healthy, unaffected embryos are available for transfer and a mock transfer has to be carried out so that such patients do not suspect that they are carriers. The  European Society of Human Reproduction and Embryology  (ESHRE) ethics task force currently recommends using  exclusion testing  instead. Exclusion testing is based on a  linkage analysis  with  polymorphic markers , in which the parental and grandparental origin of the chromosomes can be established. This way, only embryos are replaced that do not contain the chromosome derived from the affected grandparent, avoiding the need to detect the mutation itself. [ citation needed ]


Because of the sensitivity of the issue, preimplantation genetic diagnosis has sparked a rich debate in academia and beyond. [ 78 ]


Those who are against the possibility of discarding an embryo to avoid the risk of disability (such as the possible adverse selection of embryos that might develop "disabilities" such as deafness) argue that if the only two possibilities are "existence and nonexistence" ("coming into the world" or "not coming into the world"), granting them the right to come into the world is better than the alternative of not existing. [ 78 ]


Another widely used argument for rejecting preimplantation genetic diagnosis involves the meaning of the term "disability" (Bickenach & Chatterji, 2003).  The  language of medicine  describes disability as something whose functionalities deviate from a normal functioning. [ 79 ]  However, much of the disability community emphasizes that disability is often determined by the way  society structures the world . [ 80 ]  Thus, the concepts of "normalcy" and "health" are relative and depend on time, place, and society. Opponents of PGD argue against avoiding the birth of people with disabilities through preimplantation genetic diagnosis, and that people must first determine how to define what disability is. [ 81 ]


The literature highlights three most common types of arguments in favor of preimplantation genetic diagnosis. [ 78 ]
The first type of argument is mainly made by a part of academia that believes that a disability or disorder likely implies a reduced quality of life for the unborn child. [ 82 ]  The second most common type of argument focuses primarily on the duties of parents in relation to the human flourishing of the individual. This argument feeds on the idea that parents have an obligation and responsibility to ensure minimum living conditions for the unborn child. [ 82 ]


A final category of argument is one that emphasizes the importance of bringing individuals with disabilities and disorders into the world with the consideration of how much they can contribute to the socioeconomic growth of society, suggesting these individuals would not be able to contribute to improving the status quo and well-being of society as a whole. [ 78 ]


In Japan, preimplantation genetic testing-monogenic (PGT-M) has been the topic of fierce debate by individuals suffering from genetic diseases on the one hand, and feminist groups and disability activist groups on the other. As Croydon notes, "prospective PGT-M patients, such as individuals of reproductive age impacted by a disability and their spouses...have wanted to take advantage of the technology," but they have faced opposition from "groups campaigning for women's and disabled people's rights [which] include individuals who see the genetic testing of embryos in one or all of the following ways: 1) it puts undue pressure on the women involved, conveying the message to them that they are only allowed to reproduce if they are bringing into the world a healthy offspring; 2) it reinforces existing discriminatory attitudes in society towards disabled people; and 3) the circumvention of disease through the reproductive process reduces the need in the scientific community to continue the pursuit of developing treatments/cures for people who currently live with disability." Ultimately, the use of PGT-M has remained restrictive in Japan and is informally regulated by the Japan Society of Obstetrics and Gynecology (JSOG). [ 83 ]


PGD allows discrimination against those with  intersex  traits.  Georgiann Davis  argues that such discrimination fails to recognize that many people with intersex traits led full and happy lives. [ 84 ]   Morgan Carpenter  highlights the appearance of several intersex variations in a list by the  Human Fertilisation and Embryology Authority  of "serious" "genetic conditions" that may be de-selected in the UK, including  5-alpha reductase deficiency  and  androgen insensitivity syndrome , traits evident in elite women athletes and "the world's first openly  intersex mayor ". [ 85 ]   Organisation Intersex International Australia  has called for the Australian  National Health and Medical Research Council  to prohibit such interventions, noting a "close entanglement of intersex status, gender identity and sexual orientation in social understandings of sex and gender norms, and in medical and medical sociology literature". [ 86 ]


In 2015, the  Council of Europe  published an Issue Paper on  Human rights and intersex people , remarking:


Intersex people's right to life can be violated in discriminatory "sex selection" and "preimplantation genetic diagnosis, other forms of testing, and selection for particular characteristics". Such de-selection or selective abortions are incompatible with ethics and human rights standards due to the discrimination perpetrated against intersex people on the basis of their sex characteristics. [ 87 ]


PGD combined with HLA (human leukocyte antigen) matching allows couples to select for embryos that are unaffected with a genetic disease in hopes of saving an existing, affected child. The "savior sibling" would conceivably donate life-saving tissue that is compatible to his/her brother or sister. [ 88 ]  Some ethicists argue that the "savior siblings" created from this procedure would be treated as commodities. [ 88 ]  Another argument against selecting for "savior siblings" is that it leads to genetically engineered "designer babies". [ 89 ]  This argument prompts a discussion between the moral distinction of enhancing traits and preventing disease. [ 90 ]  Finally, opponents of "savior siblings" are concerned with the welfare of the child, mainly that the procedure will cause emotional and psychological harm to the child. [ 88 ]


Currently in the United States, no formal regulation or guideline exists. [ 91 ]  The ethical decisions regarding this procedure is in the discretion of health care providers and their patients. [ 91 ]  In contrast, the UK's use of PGD is regulated by the Human Fertilization and Embryology Act (HFEA), which requires clinics performing this technique to attain a license and follow strict criteria. [ 91 ]


Some religious organizations disapprove of this procedure. The Roman Catholic Church, for example, takes the position that it involves the destruction of human life. [ 92 ]  and besides that, opposes the necessary in vitro fertilization of eggs as contrary to Aristotelian principles of nature. [ citation needed ]  Orthodox Judaism, by contrast, supports PGD. [ 70 ]


A meta-analysis that was performed indicates research studies conducted in PGD underscore future research. This is due to positive attitudinal survey results, postpartum follow-up studies demonstrating no significant differences between those who had used PGD and those who conceived naturally, and ethnographic studies which confirmed that those with a previous history of negative experiences found PGD as a relief. Firstly, in the attitudinal survey, women with a history of infertility, pregnancy termination, and repeated miscarriages reported having a more positive attitude towards preimplantation genetic diagnosis. They were more accepting towards pursuing PGD. Secondly, likewise to the first attitudinal study, an ethnographic study conducted in 2004 found similar results. Couples with a history of multiple miscarriages, infertility, and an ill child, felt that preimplantation genetic diagnosis was a viable option. They also felt more relief; "those using the technology were actually motivated to not repeat pregnancy loss". [ 93 ]  In summary, although some of these studies are limited due to their retrospective nature and limited samples, the study's results indicate an overall satisfaction of participants for the use of PGD. However, the authors of the studies do indicate that these studies emphasize the need for future research such as creating a prospective design with a valid psychological scale necessary to assess the levels of stress and mood during embryonic transfer and implantation. [ 93 ]


Prior to implementing the  Assisted Human Reproduction Act  (AHR) in 2004, PGD was unregulated in Canada. The Act banned sex selection for non-medical purposes. [ 94 ]