Is it dangerous to have a baby? Worried about the baby being unhealthy? Is freezing eggs advisable? Solving the fertility problems of "older" women

The sudden gossip about celebrities in the entertainment industry yesterday has put the term "surrogacy" on the hot search, and women's fertility anxiety has been aroused once again. Since the 1970s, the Chinese government has begun to advocate late marriage and late childbearing. Today, the structure of women's childbearing age has undergone tremendous changes. Many urban women are worried about whether they are "too old" to have children, whether it will be dangerous, and whether the children will be healthy. When women face fertility problems, should they let nature take its course, or use assisted reproductive measures to "change their fate"? Today's article will answer your questions from the perspective of genetics.

Written by | Alalei (Master of Internal Medicine, Tongji University School of Medicine), Xiaorui (PhD in Obstetrics and Gynecology, Shanghai Jiao Tong University School of Medicine)

In recent years, the postponement of women's childbearing age has become a common phenomenon, especially in big cities. On the one hand, with the development of society, the social status of women has been continuously improved, the proportion of women receiving education and working outside has increased significantly, and the concept of childbearing has also changed accordingly; on the other hand, the national "two-child" policy has been fully liberalized, and more women born in the 1980s and 1990s will have a "second child" demand, and the childbearing age structure has undergone tremendous changes. At the same time, the fertility of "older" women and the health of their offspring have also attracted much attention. Our topic today starts with a real case.

Ms. Wang got pregnant twice after marriage, but both times, the baby was "gone" inexplicably within 3 months. She went to the hospital's reproductive medicine center for treatment, and her medical history showed:

Ms. Wang, 36 years old, married for 3 years, has conceived naturally twice. However, at 70 days of pregnancy, B-ultrasound showed no fetal heartbeat, and both were terminated by curettage. During the second pregnancy, chorionic villus chromosome examination was performed, and the result showed 16-trisomy syndrome (Note: it means that there is an extra chromosome 16, which leads to abnormal fetal development and spontaneous abortion). The female chromosome, male chromosome and semen examination were all within the normal range.

Ms. Wang, 36 years old, married for 3 years, has conceived naturally twice. However, at 70 days of pregnancy, B-ultrasound showed no fetal heartbeat, and both pregnancies were terminated by curettage. During the second pregnancy, chorionic villus chromosome examination was performed, and the result showed 16-trisomy syndrome (Note: it means that there is an extra chromosome 16, which can cause abnormal fetal development and spontaneous abortion). The female chromosome, male chromosome and semen examination were all within the normal range.

After a comprehensive analysis, the doctor implemented the "third-generation test tube" technology for Ms. Wang. First, the patient was given ovulation induction treatment, and the sex hormone level was affected by drugs, so that the oocytes that were originally going to degenerate could also develop and mature together, so as to obtain more mature oocytes. After obtaining the oocytes, in vitro fertilization and in vitro culture were performed, and a total of 4 normal-looking embryos were obtained. Some cells of the embryos were taken for preimplantation genetic testing, and the test results showed that 2 embryos were recommended for transplantation. Subsequently, one of the normal embryos was transplanted into the patient's uterus and a successful pregnancy was achieved.

This case can be seen as a microcosm of the fertility problems of some "older" women, which includes several key points: "36 years old", "fetal chromosome abnormalities", and "third-generation test tubes". Let's analyze what information is hidden behind these key points one by one.

It’s all about egg quality

Key point 1: Advanced age

"Older mothers" is a familiar term to everyone, and "If I don't give birth soon, I will be too old and it will affect the child" is also a concern or advice of many people. However, there is no consensus on the definition of advanced age in existing literature.

In obstetrics, advanced age is defined as a pregnant woman ≥35 years old at the time of delivery. Her fertility is significantly reduced, and the risk of fetal maldevelopment and offspring birth defects is significantly increased. A report on the impact of women's reproductive age on fertility showed that the incidence of infertility in married women aged 20-24 was 6%, 9% for those aged 25-29, 15% for those aged 30-34, 30% for those aged 35-39, and 64% for those aged 40-44.

In the reproductive field, there are different ways to distinguish. According to statistics, women reach menopause at ages 40 to 60, with an average age of 51. However, the speed of ovarian aging varies from person to person, and physiological age is not equal to ovarian age. "Advanced age" should be defined from the perspective of ovarian reserve function*, not limited to her actual age.

* There are many methods to evaluate ovarian reserve function, mainly including: clinical indicators, basal hormone levels, ovarian imaging, and ovarian stimulation experiments.

Ovarian reserve function is directly related to the number of eggs. Many people have heard that the number of eggs a woman has in her lifetime is limited, but how many are there?

A woman's eggs go through a long development process in the ovaries: primordial follicles → primary follicles → secondary follicles → mature follicles. A newborn girl has 700,000 to 2 million primordial follicles on both sides of her ovaries, about 300,000 to 500,000 at the age of 7 to 9, about 40,000 at puberty, and only a few hundred at menopause. Starting from puberty, about 15 to 20 primordial follicles begin to grow and develop at the same time each month, but usually only 1 to 2 can mature and ovulate. In the end, a woman ovulates 400 to 500 times in her lifetime, and the rest of the follicles degenerate during the development process.

However, for unexplained infertility, "advanced age" can be defined as ≥ 30 years old. For women with high-risk factors for infertility, such as single ovary, ovarian cystectomy, smoking, and a family history of premature ovarian failure (POF), "advanced age" should also be defined as ≥ 30 years old.

Key point 2: Chromosomal abnormalities

Chromosomes are the carriers of genetic material genes. Genes are passed on through cell division along with the transmission of chromosomes. If chromosomes are abnormal, such as changes in number and structure, they will cause the addition or deletion of genes, leading to chromosomal diseases. Most embryos with chromosomal abnormalities are miscarried or stillborn; patients who survive often have clinical features such as congenital multiple malformations, intellectual development disorders, and growth retardation.

We know that human reproductive cells adopt a special division method after maturity: meiosis. Meiosis means that the number of chromosomes in reproductive cells after division is halved - DNA is replicated once, and the cells divide twice in succession, and the daughter cells finally formed have only half the number of chromosomes of the mother cell.

Meiosis is roughly divided into two stages: meiosis I and meiosis II. Female egg cells (eggs) are derived from oocytes after two meiotic divisions. Their chromosomes change from 46 in oocytes (represented by 2n, called diploid) to 23 (represented by n, called haploid), and the sex chromosomes also change from XX to a single X.

In the final stage of follicular development, the mature follicle will complete the first meiotic division 36 to 48 hours before ovulation to produce a secondary oocyte. When the follicle develops into a mature follicle, it moves to the surface of the ovary, the wall of the mature follicle ruptures, and the secondary oocyte and other components are discharged from the ovary. This is ovulation.

After ovulation, the fate of the secondary oocyte is divided into two types. If it is not fertilized within 24 hours, the secondary oocyte will degenerate; if it meets and is fertilized by sperm, the secondary oocyte will complete the second meiotic division and form a mature egg cell, also known as an egg.

Ovarian aging in women can lead to decreased fertility, and the main manifestation of ovarian aging is not only a decrease in the number of oocytes, but also a decrease in cell quality. This is followed by an increase in the incidence of oocyte chromosome aneuploidy. Aneuploidy refers to an increase or decrease in the number of chromosomes in a cell by one or more. For example, if one chromosome is missing, it is called a monosomy (2n-1); if one chromosome is extra, it is called a trisomy (2n+1). The cause of oocyte aneuploidy is still unclear. Existing studies have shown [1] that during the meiosis II stage of female oocyte formation, the weakening and loss of chromosome binding force related to age, the premature occurrence of sister chromatids, and the deviation and mismatch of spindle microtubules, resulting in chromosome segregation errors, are the main causes of aneuploidy.

According to statistics, the incidence of chromosomal aneuploidy in oocytes of women aged 35 is about 10%, which rises sharply to 30% in women aged 40, 40% in women aged 43, and almost all oocytes of women over 45 are aneuploid. Therefore, with the increase of age, the incidence of chromosomal abnormalities such as Down syndrome (i.e., trisomy 21), trisomy 18, and trisomy 13, as well as the miscarriage rate, also increase accordingly. The miscarriage rate of women over 38 years old can reach 30%~40%.

Here, let's extend the topic a little. The occurrence of oocyte aneuploidy is an important factor affecting the fertility of older women. Will this situation occur in young women?

The answer is "yes".

A month ago, a study published in the American Journal of Human Genetics found that more than 7% of oocytes have at least one pair of "non-exchange" chromosomes, and this situation is not affected by maternal age. If oocytes containing non-exchange chromosomes undergo meiosis, the risk of producing aneuploid offspring is significantly increased.

"In our counseling experience, some couples who have experienced miscarriage or have had a child with an extra or missing chromosome often feel guilty," said Terry Hassold, the first author of the article, in an interview. "Our results show that the opposite is true, and many of these errors in chromosomes are inherent in human biology."

Assisted Reproductive Technology

Key point three: third-generation test tube

The third generation of test tubes, previously known as preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS), are now collectively referred to as "Preimplantation Genetic Testing" (PGT), that is, "preimplantation genetic testing". Some people may ask, since it is called "third generation", is it more advanced than the "first generation" and "second generation"? Not really! The names of the first generation (in vitro fertilization-embryo transfer, IVF-ET), the second generation (intracytoplasmic sperm injection, ICSI), and the third generation of test tubes simply represent three different assisted reproductive technologies, which are aimed at infertility caused by different reasons. There is no superiority or inferiority among the three.

The third generation of test tubes, PGT, can be divided into three categories according to different indications. The first category, PGT-A, A refers to Aneuploidies, which is suitable for the female's advanced age, unexplained repeated spontaneous abortions, unexplained repeated implantation failures, etc., which are the cases shown at the beginning of the article; the second category, PGT-SR, SR refers to Structural Rearrangements, which is suitable for either or both spouses to carry chromosomal structural abnormalities, including translocations, inversions, etc.; the third category, PGT-M, M refers to Monogenic defects, which is suitable for patients with pathogenic mutations of genetic susceptibility genes for serious diseases, and situations that require human leukocyte antigen (HLA) matching. PGT screens the embryo chromosomes and eliminates abnormal embryos, thereby reducing the risk of miscarriage and giving birth to abnormal offspring.

PGT is a complex medical and modern biotechnology project. The operation process includes:

1. Hormone-induced superovulation to obtain oocytes;

2. Use conventional in vitro fertilization or intracytoplasmic sperm injection and in vitro culture;

3. Take some embryonic cells and conduct corresponding tests using molecular biology methods;

4. Transplant the normal embryos into the uterus.

In 1968, physiologists R.L. Gardner and R.G. Edwards of Cambridge University in the United Kingdom first applied PGT technology to rabbits. They identified the sex of embryos by sex chromatin, and later determined that they were female embryos. They placed the embryos into the uterus of recipient female rabbits, and finally successfully gave birth to a batch of female rabbits[3]. In 1990, the world's first PGT applied to human embryos was completed by the A.H. Handyside team of the Department of Obstetrics and Gynecology at Hammersmith Hospital, Royal Postgraduate School of Medicine, London, UK. The treatment targets were couples carrying X-linked recessive genetic diseases. The researchers used single-cell polymerase chain reaction (PCR) to identify the sex of pre-implantation embryos, selected female embryos for transplantation, and finally successfully delivered healthy twin girls[4]. In May 2000, the Reproductive Center of the First Affiliated Hospital of Sun Yat-sen University in my country successfully performed PGT on hemophilia carriers, giving birth to the first healthy baby born in China through PGT technology[5]. Twenty years later, the Yangcheng Evening News WeChat interviewed the mother who "dared to take the lead" back then. It is understood that the girl's growth process was smooth and she was no different from other children. She was not very fond of sports but had a good memory and was currently attending university.[6]

The first case of PGT treatment was in 1990, when the molecular biology technology used was PCR. With the continuous development of science and technology, more cytogenetic technologies have been applied to PGT, greatly improving the accuracy and efficiency of PGT, such as fluorescence in situ hybridization (FISH), whole genome SNP microarray chip (SNP array), and next-generation sequencing (NGS).

It should be noted that, despite the rapid development of PGT, it is not "foolproof". On the one hand, there are currently more than 20,000 known genetic diseases in humans, and the abnormalities in chromosome number and structure vary greatly. Existing technology can only solve a small part of the problems; on the other hand, after the embryo is tested, it continues to develop and may be affected by environmental, drug and other factors, and genetic material may change again. As mentioned in the case above, patients who receive PGT treatment need to undergo amniocentesis or chorionic villus biopsy again in the second trimester to determine the genetic status of the embryo. Taking the data from the Reproductive Center of Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine as an example, the success rate of PGT transplant cycles is 60%~70%.

The key points in the case have been basically explained. Seeing this, some people may sigh: It seems that PGT is just like this! For those women who want to focus on their careers and preserve their fertility, is there a better solution? In recent years, single women in Western countries have entered the public eye for freezing their eggs. The "personal demonstration" of female stars has even made freezing eggs a "regret medicine" for preserving fertility. In December 2019, a 31-year-old unmarried woman sued the Beijing Obstetrics and Gynecology Hospital Reproductive Center in the Beijing Chaoyang District Court for refusing to freeze her eggs, which pushed freezing eggs to the forefront [7].

Egg freezing: a safe that is not safe enough

The so-called egg freezing, as the name suggests, means "freezing the egg cells." At present, the methods used for freezing oocytes mainly include slow programmed freezing and vitrification. Slow programmed freezing is an early method. The oocytes are protected by a low concentration of protective agent and slowly programmed to cool down under the control of a programmed freezer, and finally stored in liquid nitrogen. With the continuous improvement of freezing methods, programmed freezing has gradually been replaced by vitrification. The latter uses a high concentration of cryoprotectant to quickly cool the oocytes, so that the intracellular fluid is directly converted into a non-crystalline glass-like state, and finally stored in liquid nitrogen, which reduces the damage to the internal structure of the oocytes and improves the success rate.

Compared with sperm freezing (note: sperm freezing was successfully applied to artificial insemination as early as 1953) and embryo freezing, the development of oocyte cryopreservation technology is relatively slow. In 1986, Australian scientists first reported a successful pregnancy using frozen eggs (note: slow programmed freezing was used at that time) combined with in vitro fertilization technology [8]; in 2004, my country's first "frozen" (note: slow programmed freezing was used) baby was born in Peking University First Hospital [9]; in 2013, the American Society for Reproductive Medicine (ASRM) officially removed the "experimental" label of frozen eggs [10], which can be widely used in clinical practice and is regarded as "the arrival of the era of giving women reproductive rights." The specific technical, social, ethical and other issues of frozen eggs will not be discussed here. Let us focus on the genetic issues of frozen eggs.

From a genetic perspective, it was previously believed that freezing eggs is safe. However, as relevant research continues to increase, this conclusion needs to be reconsidered. In 2019, the Journal of the American Medical Association (JAMA) published a retrospective cohort study [11], led by the Danish Cancer Society Research Center, which analyzed the growth and development of 1,085,172 babies born locally between 1996 and 2012. The results showed that the risk of childhood cancer in babies born using frozen eggs was 44.4/100,000, while the risk for babies born from natural conception was 17.5/100,000. In other words, the risk of cancer in the former is more than twice that of the latter. Among them, the risk of leukemia and sympathetic nervous system tumors is significantly increased. No relevant association has been found with other assisted reproductive technologies, such as in vitro fertilization and intracytoplasmic sperm injection. In the discussion section, the authors pointed out that cryopreservation technology may cause changes in embryonic development, potentially affecting its growth in the uterus, and epigenetic changes are a possible explanation.

"Like father, like son" is our most intuitive understanding of "heredity". However, the phenomenon of "nine sons born from a dragon are all different" also implies that in addition to genes, other biological mechanisms play a role in the process of inheritance. Epigenetics refers to the various changes in gene expression without changing the DNA sequence, such as DNA modification and post-translational modification. Such changes can stabilize inheritance. However, cryopreservation technology may induce epigenetic changes in embryos, posing risks to the future growth of the baby.

In August 2020, the journal Clin Epigenetics published a review on the effects of oocyte vitrification on epigenetics and gene expression [12]. The analysis showed that in terms of epigenetic regulation, animal studies found that egg freezing had an effect on DNA methylation, miRNA, and histone modification; in terms of gene expression, human studies found that egg freezing may reduce the transcription level related to oocyte development, while animal studies showed changes in transcriptional regulation, cell differentiation and mitosis, actin cytoskeleton regulation, and apoptosis pathways. It can be seen that since the history of egg freezing technology is only more than 30 years, the genetic safety of egg freezing needs more research in the future to improve, and egg freezing is not a "fertility safe".

Summarize

In traditional concepts, people focus on "doing the right thing at the right age". However, as the pace of society accelerates, many women and their families have to face the problem of "advanced age" and turn to modern medicine for help. There are many factors that affect women's fertility, and what we will talk about today is only part of them.

Professor Peter Leung, a member of the Royal College of Science of Canada, wrote in the preface to Practical Human Assisted Reproductive Technology (edited by Huang Hefeng, People's Medical Publishing House 2018 edition): "The progress of medical knowledge and technology has brought gratifying achievements to human life and health. Assisted reproductive technology is changing with each passing day, opening a new chapter in the birth of life." However, from the perspective of doctors, we also hope to fully inform everyone of the "imperfection" of medicine. Stay objective and rational, and nature will give us the best outcome of survival of the fittest.

References

[1] Maria Shomper, Christina Lappa, and Greg FitzHarris. Kinetochore microtubule establishment is defective in oocytes from aged mice. Cell Cycle. 2014;13(7):1171-9.

[2] Terry Hassold, Heather Maylor-Hagen, Anna Wood, et al. Failure to recombine is a common feature of human oogenesis. Am J Hum Genet. 2021;108(1):16-24.

[3] RLGardner, RGEdwards. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature. 1968;218(5139):346-9.

[4] AHHandyside, EH Kontogianni,K. Hardy, et al. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344(6268):768-70.

[5] Xu Yanwen, Zhuang Guanglun, Shu Yimin, et al. Application of fluorescence in situ hybridization in preimplantation sex diagnosis. Chinese Medical Journal (English Edition). 2002;115(6):874-877.

[6] Yangcheng Evening News. 2020.07.14. China’s first third-generation test-tube baby is 20 years old this year and was born in Guangzhou!

[7] Beijing Daily Client. 2019.12.23. Woman sued hospital after hospital refused her egg freezing application.

[8] C Chen. Pregnancy after human oocyte cryopreservation. Lancet. 1986;1(8486):884-6.

[9] Li Xiaohong. Research and application of human oocyte freezing and thawing. Journal of Peking University (Medical Edition). 2004;36(6):664-667.

[10] Practice Committees of American Society for Reproductive Medicine, Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril, 2013;99(1):37-43.

[11] Marie Hargreave, Allan Jensen, Merete Kjær Hansen, et al. Association Between Fertility Treatment and Cancer Risk in Children. JAMA. 2019;322(22):2203-2210.

[12] Julie Barberet, Fatima Barry, Cecile Choux, et al. What impact does oocyte vitrification have on epigenetics and gene expression? Clin Epigenetics. 2020;12(1):121.