A Long Story Short: An Introduction To Reproductive Biology

 

Introduction: 

 

In the autumn of 2019 I completed an internship at the Department of Assisted Reproduction at Centro Médico Teknon in Barcelona, where I assisted in daily lab work and attended patient visits. This experience opened my eyes to the speciality of obstetrics and gynaecology, one that I hadn’t previously considered pursuing. Now I am truly fascinated by it. In this entry, I summarise the process that has given us life, and the measures that can be taken to help those with difficulty conceiving. 

 

 

 

Menstrual Cycle: 

 

The menstrual cycle is a change in the female reproductive system that makes pregnancy possible. The length of the cycle is 21-35 days, which explains why females that have gone through puberty experience menstrual bleeding around once a month. The first day of menstrual bleeding is considered day one of the menstrual cycle. The bleeding usually lasts around 5 days and occurs when the lining of the uterus, which has thickened to prepare for a possible pregnancy, is shed in the absence of fertilisation. The menstrual cycle is regulated by four main hormones, FSH, LH, oestrogen and progesterone, the concentrations of which vary throughout the cycle, as illustrated in the Figure. 

 

During the follicular phase, the pituitary gland produces FSH (follicle stimulating hormone), which stimulates several follicles to rise to the surface of the ovary and mature. Eventually, one follicle in one of the ovaries grows the largest and becomes the one prepared to be released at ovulation, while the other follicles shrink back. 

 

The maturing follicle produces oestrogen (estradiol), which induces the thickening of the lining of the uterus. The increased concentration of oestrogen stimulates secretion of LH (luteinising hormone) by the pituitary gland. At the end of the follicular phase, there is a large surge in the levels of LH and FSH, resulting in ovulation (egg release from the follicle). After the release, the egg enters the fallopian tube, where fertilisation can take place if sperm is present. The period of increased fertility lasts ~6 days and is known as the fertile window.  

After ovulation, the levels of FSH and LH plummet and the follicle that contained the egg transforms into a corpus luteum and begins to produce progesterone. Progesterone prevents the endometrial lining from being shed. If fertilisation does not occur, the corpus luteum disintegrates, causing progesterone levels to drop, and the endometrial lining to shed. 

 

Contraception in the form of hormone treatment (oestrogen and/or progesterone) can be used to regulate a woman’s menstrual cycle. The hormone treatments may prevent ovulation, thin the uteral wall, and thicken the cervical mucus. Progesterone has a negative feedback on LH and FSH production, resulting in the inhibition of follicular development and ovulation. Oestrogen also has a negative feedback on FSH production by the pituitary gland.  

 

 

Assisted Reproduction Technology

 

There’s a wide range of factors that may cause infertility in both men and women and it is often hard to pinpoint the exact reasons for one’s inability to conceive. In men, low sperm count or abnormal sperm function may be as a result of diabetes, genetic factors, excessive alcohol consumption and prior bacterial or viral infections. In women, hormonal disorders and structural abnormalities can affect ovulation. Structural abnormalities in the anatomy of cervix of uterus can be caused by pelvic inflammatory diseases such as chlamydia or gonorrhoea and can lead to infertility. Other factors include diabetes, high stress levels and sickle cell disease. Additionally, men and women who undergo chemotherapy also become infertile. For this reason, many patients elect to freeze ovules or sperm before undergoing chemotherapy, giving them an opportunity to use assisted reproduction technologies to have children after their treatment. Finally, assisted reproduction also gives women without a partner, as well as homosexual and asexual women, the opportunity to become mothers. 

 

 
Patient Analysis

 

Assisted reproduction usually begins with analysis of the patient’s karyotype (from a blood sample). This test evaluates the number and structure of her chromosomes in order to detect  chromosomal abnormalities (seen in ~10% of women). This test may reveal that a couple is at risk of having a child with genetic disorders, in which case an oocyte donation from a fertile, karyotypically normal woman could be considered.

 

Additionally, the blood concentration of AMH (anti-Müllerian hormone) can be measured and used as an accurate marker of female fertility. AMH is secreted by a woman’s growing follicles and can act as an indicator of potential response of the reproductive system to IVF drugs (see Infertility Medication). AMH levels decrease with age, and so too does the pregnancy rate achieved by assisted reproduction. This is around 40-50% for 35 year old patients, and increases to ~60% when using oocytes from a fertile donor (bare in mind that these women may already have problems with their reproductive system). For women above 40 it is highly recommended that donor oocytes are used in IVF. 

 

 

Sperm Analysis

 

The semen of the partner (or donor) is then analysed in what is known as a seminogram. This enables assessment of the quality of the sperm sample. The process is outlined below: 

• Liquify and make the sample more homogenous by incubating it at 37°C for 5 minutes
• Measure ejaculate volume (< 1.5 ml is abnormal) 
• Measure semen viscosity (by measuring the length of a drop generated using a plastic pipette, where > 2 cm drops are abnormal)  
• Measure semen pH (usually 7.2 - 7.5; an acidic pH may indicate blockage of seminal vesicles, while a basic pH may indicate an infection)

 

The sperm concentration can be measured under a microscope using a Haemocytometer. A diluted semen sample is used, and the lower reference limit is around 15 million spermatozoa/ml. A low sperm count is known as azoospermia. 

 

Then, the sperm motility can be calculated. Sperm can have progressive motility (active movement linearly or in a large circle), non-progressive motility (all other patterns of motion), or be immobile. The lower limit for progressive motility is around 33%. 

 

Next, the percentage sperm vitality can be measured using with an eosin stain, which increases the contrast between the background and the sperm heads. The sperm heads will appear dark in membrane-damaged cells, while live spermatozoa will have white or light pink heads. The lower reference limit for vitality is ~58%

 

Leukocytes and other white blood cells are present in most human ejaculates. These can cause infertility by damaging spermatozoa and their genetic material. Although leukocytes are round, they may be confused for aggregations of sperm cells under the microscope. Instead, they can be identified by a peroxidase-staining test. 

 

The presence of IgG and IgA antibodies in semen may cause agglutination of spermatozoa, reducing their ability to swim, also causing infertility. The most common antibody test is the mixed anti globulin reaction (MAR) test.

 

Lastly, DNA can be extracted from the sperm cells for Fluorescence In Situ Hybridisation (FISH) analysis. FISH is a technique that uses fluorescent probes that bind only those parts of a nucleic acid sequence with a high degree of sequence complementarity. This can indicate whether the  patient karyotype is normal or not. Using spermatozoa with an altered FISH can reduce the chances of achieving a successful pregnancy and increase the likelihood of aneuploid embryos. Using these analyses, it can be assessed whether the semen sample is viable for use in assisted reproduction, or whether a sperm donor may be required. 

 

When the patient is unable to generate an ejaculate, such as in the case of impotence, a testicular biopsy under local anaesthetic may be used to retrieve spermatozoa from the testicular tubes. The spermatozoa are then used to fertilise oocytes by intracellular sperm injection (ICSI - see later). However, these sperm  cells tend to be more immature than those found in in ejaculates and usually have a lower chance of fertilisation and generate lower quality embryos. Testicular biopsies are also used in patients with a vasectomy. This is a surgical male sterilisation procedure that stops spermatozoa getting into a man’s semen by permanently sealing the tubes that carry a sperm. 

 

 

Artificial Insemination 

 

After completing a seminogram, the sample is centrifuged in two steps to obtain live, progressively motile spermatozoa. A high viscosity medium is used, which allows only rapidly moving spermatozoa to swim towards the pellet. This pellet is resuspended in suitable media for artificial insemination (AI). During AI, the sample is introduced into a female’s cervix or uterine cavity in a process of in vivo fertilisation without sexual intercourse. This is usually attempted when the semen sample is good and it achieves a pregnancy rate of ~10%.

 

In Vitro Reproduction 

 

IVF has become a well known and commonly understood term in recent years. In this process, the sperm and eggs are combined on a laboratory dish and left to fertilise, known as insemination. Nowadays, IVF is often replaced by an alternative in vitro fertilisation technique known as ICSI (intracytoplasmic sperm injection). In this procedure, a single sperm cells is injected directly into the cytoplasm of the egg. ICSI only needs a single sperm cell per oocyte, while IVF requires fifty to hundred thousand. Additionally ICSI is extremely fast; an oocyte can be fertilised in a minute. IVF can take several days and has a lower probability of fertilisation. Nonetheless, ICSI remains a more invasive technique and is avoided when there is a rich semen sample and healthy oocytes. Despite their differences, the steps leading up to insemination are the same for classic IVF and ICSI: 

 

 

Infertility Medication 

 

Before the retrieval of a patients oocytes, they must undergo an infertility treatment for ovarian stimulation. First, oral medications are taken to stimulate FSH secretion by the pituitary gland. Additionally, the FSH hormone itself is injected subcutaneously (under the skin) to bypass the hypothalamus and pituitary glands and directly stimulate follicle growth in the ovaries. Infertility medication can have potent side effects including depression, insomnia, headaches and nausea. Thus, it is essential that these medicines are well administered and controlled. 

 

 

Transvaginal Oocyte Retrieval 

 

Oocyte retrieval is carried out under general anaesthetic. Under ultrasound guidance, the gynaecologist inserts a needle through the vaginal wall and into each individual ovarian follicle. The other end of the needle is attached to a suction device, which aspirates the follicular fluid, hopefully containing an oocyte. The follicular fluid is yellow-red in appearance. The presence of blood in the aspirated sample causes a dark red appearance and is a sign of endometriosis, a painful disorder where the tissue that normally lines the uterus also grows outside the uterus. Because patients are hyper stimulated by hormonal treatments, it is common to retrieve ~15 oocytes. The procedure is carried out just prior to ovulation. The oocytes obtained are then quantified and analysed. 

 

To check whether the oocytes are mature, their exterior cell mass is removed chemically (in a medium containing enzymes) and physically, by pipetting up and down. A mature oocyte can be identified by the presence of a polar corpuscle, found outside the cell. This means that the oocyte has undergone two rounds meiosis and therefore contains half the genetic material. During prophase I, the egg contains a visible nucleus. This disappears in metaphase I, when the nuclear membrane disintegrates. In metaphase II, the nucleus is not visible and the polar corpuscle has moved to the outside of the cell. The presence of aggregations of endoplasmic reticulum in the egg can be detected under the microscope. This signals high lipid production, which indicates that the egg may be in a way ‘overwhelmed’. On the same day of retrieval, the eggs can be fertilised by IVF or ICSI. The resulting embryos are cultured in a special incubator until they reach their blastocyst stage at ~day 5.  

 

Embryonic development 

 

Upon the fusion of gametes, the fertilised egg membrane rapidly depolarises to ensure the egg is fertilised by a single sperm. Then, cell division produces a cluster of cells that is the same size as the original zygote, known as cleavage. After four divisions, 3-4 days post fertilisation, a dense ball of 16 totipotent cells know as blastomeres is formed, called a morula. Around 5 days after fertilisation, the morula develops into a blastocyst. A blastocyst has a cavity inside the zona pellucida along with an inner cell mass. The outer layer consists of cells collectively known as the trophoblast. It comprises about 200-300 cells. At the blastocyst stage, the embryos can be frozen and stored until the day of the transfer. 

 

Before transfer, some patients elect to receive PGS (preimplantation genetic screening). PGS testing is a genetic study in which blastomeres are biopsied out of a blastocyst and are analysed. This procedure enables distinction between euploid, mosaic, and aneuploid cells embryos. Euploid human embryos contain 46 chromosomes per cell, while aneuploid embryos have chromosomal abnormalities. Mosaic embryos contain cells with normal karyotypes, as well as cells with altered karyotypes. Mosaic embryos may also be transferred, always whenever they don’t contain chromosomal abnormalities that could result in developmental disorders (e.g. chromosome 21 triploidy causes Down’s Syndrome). A mosaic embryo can lead to a successful pregnancy of a healthy baby when the aneuploid cells die during development, while the euploid cells divide. PGS is recommended by the American College of Gynaecology and Obstetrics to women over 35 who are receiving assisted reproduction therapy.

 

 

Transfer

 

Beginning two days after oocyte retrieval, progesterone is administered through a vaginal gel or a pill. This prevents the endometrial lining from being shed. The embryos are thawed on the morning prior to the embryonic transfer. 

 

The embryo transfer procedure begins with placing a speculum in the vagina to visualise the cervix, which is cleansed with saline solution. A hard catheter is first used to generate a channel into the neck of the uterus, after which a soft catheter loaded with the embryo is advanced into the uteral cavity. The embryo is released into this cavity. To facilitate entry, it is advised that patients attend their transfer with a full bladder, which straightens up the uterus. Following the transfer, vaginal application of progesterone will continue for 9 days. At this point, the patients will visit their doctor to carry out a pregnancy test. 

 

 

Pregnancy

 

Pregnancy lasts about 40 weeks, counting from the first day of a woman’s last normal period. The weeks are arranged into three trimesters. During the first trimester, the body undergoes hormonal changes that affect almost every organ in your body. Menstrual bleeding stops and the body can experience extreme tiredness, mood swings, an upset stomach (morning sickness) and other symptoms.

 

In the second trimester, many discomfort symptoms can go away. Towards the end of the second semester (week 28) you can feel your baby moving! Body changes during the second semester may include body aches, stretch marks on the abdomen and breast and itching in the abdomen, hands and feet. Loss of appetite, nausea and vomiting in this trimester may be the result of a liver problem, thus it is important to keep your doctor informed. 

 

In the third trimester, many women may develop breathing difficulties due to the fact that the growing baby is putting pressure on the organs. As women near the due date their cervix becomes thinner and softer, which will allow the birth canal to open during birth. 

  

Childbirth 

 

Babies can enter this world by either vaginal birth or by a surgical delivery by Caesarean section. A C-section involves opening up a pregnant woman’s abdomen and removing the baby from her uterus. This procedure requires epidural administration of anaesthesia (when anaesthetic is injected into the epidural space of the spinal cord). 

 

A C-section may be planned for medical reasons that make vaginal birth too risky, for example, if the woman is expecting twins, or if she suffers from a medical condition such as diabetes or high blood pressure. Additionally, if the mother suffers from HIV or genital herpes, a C-section may ensure that the infection is not passed on to her baby during birth. C-sections may also be necessary when a mother is delivering a large baby but has a small pelvis.

 

Sometimes, an emergency C-section is performed when the health of the baby or mother are in jeopardy, such as if the baby is not receiving sufficient oxygen during labour. This is a common complication of breech birth, which is when the baby is oriented ‘bottom first’ for a vaginal delivery. Breech birth occurs in 3-5% of women, where foetal head entrapment and umbilical cord collapses are the major risks causing oxygen deprivation for the baby. 

 

Although C-sections are considered safe, they may carry additional risks compared to vaginal delivery. The procedure results in increased blood loss and risk of infection for the mother, making women three times more likely to die than during vaginal delivery. Vaginal delivery may also have health benefits for the baby; an early exposure to bacteria as the baby passes through the birth canal may boost its immune system. Additionally, vaginal birth can squeeze out fluid from the newborn’s lungs, reducing the amount of breathing difficulties (e.g. asthma) experienced compared to Caesarean babies.  

 

The disadvantages of vaginal delivery are that, if the labour is long, there is an increased risk of the baby getting injured during birth, such as fractured collarbone or a bruised scalp. However, the hospital stay for a normal vaginal birth tends to 24-48 hours. Vaginal delivery is often assisted using forceps, which clamp onto the baby’s head and guide it out of the birth canal. 

 

 

Ectopic Pregnancy and Abortion 

 

An ectopic pregnancy is a complication of pregnancy in which the embryo implants itself outside the uterus, usually in one fo the fallopian tubes. The signs and symptoms classically include vaginal bleeding and abdominal pain. Unfortunately it not possible to safe an ectopic pregnancy and it usually has to be removed by medication or an operation. 

 

The process of ending a pregnancy is called an abortion or termination. The pregnancy is ended either by taking medications or having a minor surgical procedure, which involves general or local anaesthetic. One in three women have an abortion in their lifetime. In England, Wales and Scotland, abortions can be carried out before 24 weeks of pregnancy and even later in cases where the mother’s life is at risk or the child could be born with severe disability.  24 weeks is the threshold at which premature babies are more likely to survive. In Spain, abortions take place in the first trimester (up to 13 weeks) and in the second trimester when the mother or foetus are at serious risk.